Specific Binding Members For Ngf

ABSTRACT

Specific binding members for Nerve Growth Factor (NGF), in particular anti-NGF antibody molecules, especially human antibody molecules, and especially those that neutralise NGF activity. Methods for using anti-NGF antibody molecules in diagnosis or treatment of NGF related disorders, including pain, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, other diseases of airway inflammation, diabetic neuropathy, cardiac arrhythmias, HIV, arthritis, psoriasis and cancer.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A DISKETTE

A diskette copy of the Sequence Listing of Sequences 1 to 537 which issaved as sequence listing.txt and is 253 KB, and is submitted herewithand is incorporated by reference herein in its entirety for allpurposes.

The present invention relates to specific binding members, in particularanti-NGF antibody molecules, especially human antibody molecules, andespecially those that neutralise NGF (Nerve Growth Factor) activity. Itfurther relates to methods for using anti-NGF antibody molecules indiagnosis or treatment of NGF related disorders, including pain, asthma,chronic obstructive pulmonary disease, pulmonary fibrosis, otherdiseases of airway inflammation, diabetic neuropathy, cardiacarrhythmias, HIV, arthritis, psoriasis and cancer.

The present invention provides antibody molecules of particular value inbinding and neutralising NGF, and thus of use in any of a variety oftherapeutic treatments, as indicated by the experimentation containedherein and further by the supporting technical literature.

Nerve growth factor (β-NGF, commonly known as NGF) plays a well-knownpivotal role in the development of the nervous system. In the adult,however, NGF plays a more restricted role, where it promotes the healthand survival of a subset of central and peripheral neurons (Huang &Reichardt, 2001). NGF also contributes to the modulation of thefunctional characteristics of these neurons. As part of this latterprocess, NGF exerts tonic control over the sensitivity, or excitability,of nociceptors (Priestley et al., 2002; Bennett, 2001). These peripheralneurons sense and transmit to the central nervous system the variousnoxious stimuli that ultimately give rise to perceptions of pain(nociception). Thus, agents that reduce levels of NGF may possessutility as analgesic therapeutics.

The societal cost of inadequately treated pain further supports thepotential utility of analgesics based on anti-NGF activity. That is,despite the existence and widespread use of numerous pain medications, aclear need exists for new analgesics. Pain is one of the most commonsymptoms for which medical assistance is sought and is the primarycomplaint of half of all patients visiting a physician. The high cost ofpain to society is well documented. In the U.S., for example, chronicpain afflicts some 34 million Americans. Pain results in 50 millionworkdays lost each year. Direct medical costs attributed to back pain,arthritic pain, and migraine amount to $40 billion annually alone. Thetotal prescription pain medication market is approximately $15 billionper year (Pleuvry & Pleuvry).

As these statistics imply, a substantial percentage of pain sufferersfail to receive adequate pain relief. As a consequence, a large medicalneed remains for safe and effective analgesics with novel mechanisms ofaction (Pleuvry & Pleuvry).

Therapeutic agents that reduce the tissue levels or inhibit the effectsof secreted NGF have the potential to be just such novel analgesics.Subcutaneous injections of NGF itself produce pain in humans andanimals. Thus, injected NGF causes a rapid thermal hyperalgesia,followed by delayed thermal hyperalgesia and mechanical allodynia (Pettyet al., 1994; McArthur et al., 2000). Endogenously secreted NGF issimilarly pro-nociceptive. Tissue-injury-induced release of NGF and itssubsequent action in the periphery plays a major role in the inductionof thermal hyperalgesia through the process of ‘peripheralsensitization’ (Mendell & Arvanian, 2002). Tissue injury promotes therelease of pro-nociceptive and pro-inflammatory cytokines, which, inturn, induce the release of NGF from keratinocytes and fibroblasts. Thisreleased NGF acts directly on nociceptors to induce painful ornociceptive states within minutes of the noxious insult. This NGF alsoacts indirectly to induce and maintain nociceptive/pain states. Ittriggers mast cell degranulation, releasing pro-nociceptive agents suchas histamine and serotonin and, importantly, more NGF, and can alsostimulate sympathetic nerve terminals to release pro-nociceptiveneurotransmitters, such as noradrenaline (Ma & Woolf, 1997).

Tissue levels of NGF are elevated in CFA- and carrageenan-injectedanimals (Ma & Woolf, 1997; Amann & Schuligoi, 2000). Moreover, increasedlevels of NGF have been documented in patients suffering from rheumatoidarthritis (Aloe & Tuveri, 1997) or cystitis (Lowe et al., 1997). Inrodents, peripheral nerve injury increases the expression of NGF mRNA inmacrophages, fibroblasts, and Schwann cells (Heumann et al., 1987).Over-expression of NGF in transgenic mice results in enhancedneuropathic pain behavior following nerve injury above that of wild-typemice (Ramer et al., 1998). Over hours and days, elevated NGF levels playa role in promoting ‘central sensitization’—the enhancement ofneurotransmission at synapses in the nociceptive pathways of the spinalcord. Central sensitization results in persistent and chronichyperalgesia and allodynia. This process is thought to involveinternalization of complexes of NGF and its high affinity receptor, trkA(tyrosine receptor kinase A). Retrograde transport of these complexes tonociceptor cell bodies in the dorsal root ganglia (DRG) potentiatessecretion of nociceptive neuropeptides (e.g., substance P, CGRP), PKCactivation, and NMDA receptor activation in the dorsal horn of thespinal cord (Sah et al., 2003)—all processes that promote thesensitization of the nociceptive pathways. NGF also plays a role in theup-regulation and re-distribution of voltage-dependent and ligand-gatedion channels, including sodium channel subtypes and the capsaicinreceptor, VR1 (Mamet et al., 1999; Fjell et al., 1999; Priestley et al.,2002). The altered activities and/or expression of transmitters,receptors, and ion channels underlie the increased sensitivity andexcitability of nociceptors associated with neuropathic pain states.

NGF can also promote the sprouting of sympathetic neurons and theformation of aberrant innervation of nociceptive neurons. Thisinnervation is thought to contribute to the induction and maintenance ofchronic nociceptive/pain states, such as sympathetically maintainedpain, or complex regional pain syndrome (Ramer et al., 1999).

NGF-induced nociception/pain is mediated by the high affinity NGFreceptor, trkA (tyrosine receptor kinase A) (Sah, et al., 2003). About40-45% of nociceptor cell bodies in DRGs express trkA. These are thecell bodies of the small diameter fibers, or C-fibers, that also expressthe secreted pro-nociceptive peptides, substance P and CGRP. Thesefibers terminate in laminae I and II of the dorsal horn, where theytransfer to the central nervous system the noxious stimuli sensed byperipheral nociceptors. Mutations or deletions in the trkA gene producea phenotype characterized by loss of pain sensation both in humans(Indo, 2002) and in trkA knock-out mice (de Castro et al., 1998).Significantly, the expression of trkA is up-regulated in animalssubjected to models of arthritic (Pozza et al., 2000) or cystitic pain(Qiao & Vizzard, 2002), or the inflammatory pain induced by injection ofcomplete Freund's adjuvant (CFA) or carrageenan into the paw (Cho etal., 1996).

NGF also binds to the p75 neurotrophin receptor. The role of the p75receptor is dependent on its cellular environment and the presence ofother receptors with which it is believed to play an accessory orco-receptor function. Interaction between the trkA and p75 receptorsresults in the formation of high affinity binding sites for NGF. Theimportance of such receptor interactions in NGF-mediated pain signallingis not clear, but recent studies have implicated the p75 receptor incellular processes that may be relevant (Zhang & Nicol, 2004). However,whilst p75 receptor knockout mice display elevated thresholds to noxiousstimuli, they remain responsive to the hyperalgesic effects of NGF,suggesting that trkA receptors alone are sufficient to mediate theseeffects (Bergmann et al., 1998).

The evidence cited above indicates that NGF-mediated processes areresponsible for the induction of acute pain, short-term pain, persistentnociceptive pain, and persistent or chronic neuropathic pain. Thus,anti-NGF agents are indicated as having utility as effective analgesicsfor treating sufferers of any or all of these various pain states.

One such anti-NGF agent is trkA-Fc, which acts as a decoy or scavengerto bind up, and thereby inactivate, endogenous NGF. TrkA-Fc is a fusionprotein consisting of the NGF binding region of trkA linked to aconstant domain fragment (Fc) of an IgG antibody. TrkA-Fc produceshypoalgesia in naïve animals, decreases nociceptor responses, anddecreases sprouting of unmyelinated pain-sensing neurons (Bennett etal., 1998).

Antisera raised against NGF can also reduce NGF levels when injectedlocally or systemically. Both anti-NGF antisera and trkA-Fc attenuatecarrageenan- or CFA-induced inflammatory paw pain (Koltzenberg et al.,1999) and inflamed bladder responses in rats (Jaggar et al., 1999).Anti-NGF antiserum blocks heat and cold hyperalgesia, reversesestablished thermal hyperalgesia, and prevents collateral sprouting inthe chronic constriction injury (CCl) model of neuropathic pain (Woolf,1996; Ro et al., 1999). Small molecule inhibitors of the trkA-NGFinteraction have also been reported. In rats, the NGF-trkA inhibitorALE-0540 reduces hyperalgesia in a thermally-induced inflammatory painmodel and in the formalin test of acute and persistent pain (Owolabi etal., 1999). ALE-0540 also reduces mechanical allodynia in the sciaticnerve injury model of neuropathic pain (Owolabi et al., 1999).

Therapeutic antibodies in general hold out the promise of a degree oftarget selectivity within a family of closely related receptors,receptor ligands, channels, or enzymes that is rarely attainable withsmall molecule drugs. NGF-mediated pain is particularly well suited tosafe and effective treatment with antibodies because NGF levels increasein the periphery in response to noxious stimuli and antibodies have lowblood-brain barrier permeability. Whilst polyclonal antibodies have beenshown to be effective in animal models of pain, anti-NGF monoclonalantibodies are more likely to be successfully developed as humantherapeutics due to the advantages in manufacturing and characterizing aconsistent, well-defined, chemical, entity. The anti-nociceptive effectsof mouse anti-NGF monoclonal antibodies (Sammons et al., 2000) have beenreported, but the amino acid sequences of these antibodies were notprovided.

Recent evidence suggests that NGF promotes other pathologies in additionto pain. Thus, anti-NGF antibodies may also possess utility for treatingother NGF-mediated diseases, including but not limited to asthma,chronic obstructive pulmonary disease, pulmonary fibrosis, otherdiseases of airway inflammation (Hoyle, 2003; Lommatzch et al., 2003),diabetic neuropathy (Yasuda et al., 2003), cardiac arrhythmias(WO04/032852), HIV (Garaci et al., 2003), arthritis, psoriasis andcancer (Nakagawara, 2001).

WO02/096458 relates to anti-NGF antibodies, in particular mousemonoclonal antibody 911, and use of such antibodies in treatment ofvarious NGF-related disorders, including pain, asthma, arthritis andpsoriasis. It states that the antibody 911 had no adverse effect on theimmune system in an experimental mouse model of allergy. Theseantibodies were also described by Hongo et al., 2000.

WO04/032870 describes the pain-reducing effect of the mouse monoclonalNGF antibody mab 911 and humanized NGF antibody E3 in experimentalmodels of post-operative pain. E3 differs from human heavy chain gamma2aconstant region by 2 amino acids.

WO04/032852 describes methods for preventing sudden cardiac death andfor treatment of cardiac arryhthmias using NGF antagonists.

WO 01/78698 describes the use of polyclonal antiserum to NGF to treatchronic visceral pain.

The present invention provides specific binding members for NGF,preferably human NGF. Thus, a specific binding member of the inventionmay bind human NGF or non-human NGF (e.g. non-human primate NGF and/orrat NGF and/or mouse NGF).

Specific binding members of the invention may be antibodies to humanNGF, especially human antibodies, which may be cross-reactive withnon-human NGF, including non-human primate NGF and/or mouse NGF and/orrat NGF.

A specific binding member in accordance with the present inventionpreferably neutralises NGF. Neutralisation means reduction or inhibitionof biological activity of NGF, e.g. reduction or inhibition of NGFbinding to one or more of its receptors (preferably TrkA). The reductionin biological activity may be partial or total. The degree to which anantibody neutralises NGF is referred to as its neutralising potency.Potency may be determined or measured using one or more assays known tothe skilled person and/or as described or referred to herein, forexample:

-   -   “FLIPR” calcium mobilisation assay (see Example 2 herein)    -   PC12 survival assay (see Example 5 herein)    -   TF-1 proliferation assay (see Example 6 herein)    -   Receptor binding inhibition assay (see Example 9 herein). Assays        and potencies are described in more detail elsewhere herein.

Specific binding members of the present invention may be optimised forneutralising potency. Generally potency optimisation involves mutatingthe sequence of a selected specific binding member (normally thevariable domain sequence of an antibody) to generate a library ofspecific binding members, which are then assayed for potency and themore potent specific binding members are selected. Thus selected“potency-optimised” specific binding members tend to have a higherpotency than the specific binding member from which the library wasgenerated. Nevertheless, high potency specific binding members may alsobe obtained without optimisation, for example a high potency specificbinding member may be obtained directly from an initial screen e.g. abiochemical neutralisation assay. The present invention provides bothpotency-optimised and non-optimised specific binding members, as well asmethods for potency optimisation from a selected specific bindingmember. The present invention thus allows the skilled person to generatespecific binding members having high potency.

A specific binding member in accordance with the present inventionpreferably exhibits antihyperalgesic and/or antiallodynic activity, e.g.inhibits carrageenan-induced thermal hyperalgesia.

In some embodiments, a specific binding member of the inventioncomprises an antibody molecule. In other embodiments, a specific bindingmember of the invention comprises an antigen-binding site within anon-antibody molecule, e.g. a set of CDRs in a non-antibody proteinscaffold, as discussed further below.

In various aspects and embodiments of the invention there is providedthe subject-matter of the claims included below.

Preferred embodiments within the present invention are antibodymolecules, whether whole antibody (e.g. IgG, such as IgG4) or antibodyfragments (e.g. scFv, Fab, dAb). Preferably, an antibody molecule of theinvention is a human antibody molecule. Antibody molecules comprisingantibody antigen-binding sites are provided, as are antibody VH and VLdomains. Within VH and VL domains are provided complementaritydetermining regions, (“CDRs”), and framework regions, (“FRs”), to formVH or VL domains as the case may be. An antibody antigen-binding sitemay consist of an antibody VH domain and/or a VL domain. All VH and VLsequences, CDR sequences, sets of CDRs and sets of HCDRs and sets ofLCDRs disclosed herein represent aspects and embodiments of theinvention. A “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set ofHCDRs means HCDR1, HCDR2 and HCDR3, and a set of LCDRs means LCDR1,LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs” includes HCDRsand LCDRs.

Examples of antibody VH and VL domains and CDRs according to the presentinvention are as listed in the appended sequence listing.

A number of antibody lineages are disclosed herein, defined withreference to sequences, e.g. a set of CDR sequences, optionally with oneor more, e.g. one or two, or two substitutions. The preferred parentlineage is the 1021E5 lineage. The 1021E5 lineage includes the preferredantibody molecule 1133C11 and other antibody molecules of the “1133C11lineage”, including 1252A5. Also within the 1021E5 parent lineage areantibody molecules 1165D4, 1230H7 and 1152H5. The present inventors haveidentified the 1021E5, 1083H4 and especially the 1133C11 lineages asproviding human antibody antigen-binding sites against NGF that are ofparticular value.

The 1133C11 lineage is defined with reference to a set of six CDRsequences of 1133C11 as follows: HCDR1 SEQ ID NO: 193, HCDR2 SEQ ID NO:194, HCDR3 SEQ ID NO: 195, LCDR1 SEQ ID NO: 198, LCDR2 SEQ ID NO: 199,and LCDR3 SEQ ID NO: 200. The set of CDRs wherein the HCDR1 has theamino acid sequence of SEQ ID NO: 193, the HCDR2 has the amino acidsequence of SEQ ID NO: 194, the HCDR3 has the amino acid sequence of SEQID NO: 195, the LCDR1 has the amino acid sequence of SEQ ID NO: 198, theLCDR2 has the amino acid sequence of SEQ ID NO: 199, and the LCDR3 hasthe amino acid sequence of SEQ ID NO: 200, are herein referred to as the“1133C11 set of CDRs”. The HCDR1, HCDR2 and HCDR3 within the 1133C11 setof CDRs are referred to as the “1133C11 set of HCDRs” and the LCDR1,LCDR2 and LCDR3 within the 1133C11 set of CDRs are referred to as the“1133C11 set of LCDRs”. A set of CDRs with the 1133C11 set of CDRs,1133C11 set of HCDRs or 1133C11 LCDRs, or one or two substitutionstherein, is said to be of the 1133C11 lineage.

Other preferred lineages and sets of CDRs are defined with reference tothe analogous CDRs as set out anywhere herein, including as referredembodiments the sets of CDRs disclosed in Table 2a (with SEQ ID NOS asset out in Table 2b). Table 2a and Table 2b show sets of CDRs (HCDRs andLCDRs) from optimised clones derived from clone 1021E5, illustrating howthe CDR sequences of the optimised clones differ from those of 1021E5. Aset of CDRs from Table 2a/2b includes a set of HCDRs and/or a set ofLCDRs from any clone illustrated in the Table, optionally including1021E5 itself.

Sets of CDRs of these are provided, as indicated, as are sets of CDRswith the disclosed sequences containing one or two amino acidsubstitutions.

The present invention also provides specific binding members andantibody molecules comprising the defined sets of CDRs, set of HCDRs orset of LCDRs, as disclosed herein, and sets of CDRs of with one or twosubstitutions within the disclosed set of CDRs. The relevant set of CDRsis provided within an antibody framework or other protein scaffold, e.g.fibronectin or cytochrome B (Koide et al., 1998; Nygren et al., 1997),as discussed below. Preferably antibody framework regions are employed.For example, one or more CDRs or a set of CDRs of an antibody may begrafted into a framework (e.g. human framework) to provide an antibodymolecule or different antibody molecules. For example, an antibodymolecule may comprise CDRs of an antibody of the 1021E5 lineage andframework regions of human germline gene segment sequences. An antibodyof a lineage may be provided with a set of CDRs within a framework whichmay be subject to “germlining”, where one or more residues within theframework are changed to match the residues at the equivalent positionin the most similar human germline framework (e.g. DP10 from the VH1family) or a framework of the λ1 family e.g. DPL5. Thus, antibodyframework regions are preferably germline and/or human.

The invention provides an isolated human antibody specific for NGF,having a VH domain comprising a set of HCDRs in a human germlineframework comprising DP10. Normally the specific binding member also hasa VL domain comprising a set of LCDRs, preferably in a human germlineframework comprising a Vλ1, e.g. DPL5. Preferably, the CDRs are a set ofCDRs disclosed herein.

By “substantially as set out” it is meant that the relevant CDR or VH orVL domain of the invention will be either identical or highly similar tothe specified regions of which the sequence is set out herein. By“highly similar” it is contemplated that from 1 to 5, preferably from 1to 4 such as 1 to 3 or 1 or 2, or 3 or 4, amino acid substitutions maybe made in the CDR and/or VH or VL domain.

In one aspect, the present invention provides a specific binding memberfor NGF, comprising an antibody antigen-binding site which is composedof a human antibody VH domain and a human antibody VL domain and whichcomprises a set of CDRs, wherein the VH domain comprises HCDR1, HCDR2and HCDR3 and the VL domain comprises LCDR1, LCDR2 and LCDR3, whereinthe HCDR1 has the amino acid sequence of SEQ ID NO: 193, the HCDR2 hasthe amino acid sequence of SEQ ID NO: 194, the HCDR3 has the amino acidsequence of SEQ ID NO: 195, the LCDR1 has the amino acid sequence of SEQID NO: 198, the LCDR2 has the amino acid sequence of SEQ ID NO: 199, andthe LCDR3 has the amino acid sequence of SEQ ID NO: 200; or wherein theset of CDRs contains one or two amino acid substitutions compared withthis set of CDRs.

Thus, the invention provides a specific binding member for NGF,comprising an antibody antigen-binding site which is composed of a humanantibody VH domain and a human antibody VL domain and which comprises aset of CDRs, wherein the set of CDRs is the 1133C11 set of CDRs or otherset of CDRs disclosed herein, or a set of CDRs containing one or twosubstitutions compared with the 1133C11 set of CDRs or other set of CDRsdisclosed herein.

In preferred embodiments, the one or two substitutions are at one or twoof the following residues within the CDRs of the VH and/or VL domains,using the standard numbering of Kabat (1991).

31, 34 in HCDR1

51, 55, 56, 57, 58, 65 in HCDR2

96 in HCDR3

26, 27, 27A, 27B, 28, 29, 30 in LCDR1

56 in LCDR2

90, 94 in LCDR3.

In preferred embodiments one or two substitutions are made at one or twoof the following residues within the 1133C11 set of CDRs in accordancewith the identified groups of possible substitute residues: SubstituteResidue Position of selected from the group substitution consisting of31 in HCDR1: A 34 in HCDR1: V 51 in HCDR2: V 55 in HCDR2: N 56 in HCDR2:A 57 in HCDR2: V 58 in HCDR2: S 65 in HCDR2: D 96 in HCDR3: N 26 inLCDR1: T 26 in LCDR1: G 27 in LCDR1: N 27 in LCDR1: R 27A in LCDR1: T27A in LCDR1: P 27B in LCDR1: D 28 in LCDR1: T 29 in LCDR1: E 30 inLCDR1: D 56 in LCDR2: T 90 in LCDR3: A 94 in LCDR3: G.

Residue 29E within LCDR1 is a particularly preferred embodiment.

Preferred embodiments have the 1133C11 or 1252A5, 1152H5, 1165D4, 1230H7or 1021E5 set of CDRs.

In one embodiment an isolated specific binding member comprises a set ofCDRs which contains the 1133C11 set of CDRs with the amino acid sequenceFNSALIS (SEQ ID NO: 532) or the amino acid sequence MISSLQP (SEQ ID NO:533), substituted for the amino acid sequence LNPSLTA (SEQ ID NO: 531)within HCDR3.

Any set of HCDRs of the lineages disclosed herein can be provided in aVH domain that is used as a specific binding member alone or incombination with a VL domain. A VH domain may be provided with a set ofHCDRs of a 1133C11, 1021E5 or other lineage antibody, e.g. a set ofHCDRs as illustrated in Table 2a/2b, and if such a VH domain is pairedwith a VL domain, then the VL domain may be provided with a set of LCDRsof a 1133C11, 1021E5 or other lineage antibody, e.g. a set of LCDRs asillustrated in Table 2a/2b. A pairing of a set of HCDRs and a set ofLCDRs may be as shown in Table 2a/2b, providing an antibodyantigen-binding site comprising a set of CDRs as shown in Table 2a/2b.

The VH and VL domain frameworks comprise framework regions, one or moreof which may be a germlined framework region, normally human germline.The VH domain framework is preferably human heavy chain germ-lineframework and the VL domain framework is preferably human light chaingerm-line framework. Framework regions of the heavy chain domain may beselected from the VH-1 family, and a preferred VH-1 framework is a DP-10framework. Framework regions of the light chain may be selected from theλ1 family, and a preferred framework is DPL5.

One or more CDRs may be taken from the 1252A5 VH or VL domain andincorporated into a suitable framework. This is discussed furtherherein. 1252A5 HCDRs 1, 2 and 3 are shown in SEQ ID NO: 393, 394, 395respectively. 1252A5 LCDRs 1, 2 and 3 are shown in SEQ ID NO: 398, 399,400, respectively.

All this applies the same for other CDRs and sets of CDRs as disclosedherein, especially for 1152H5, 1165D4 and 1230H7.

Embodiments of the present invention employ the antibody VH and/or VLdomain of an antibody molecule of the 1021E5 lineage, e.g. the antibodymolecule 1021E5. A specific binding member comprising an antibodyantigen-binding site comprising such a VH and/or VL domain is alsoprovided by the present invention.

Preferred embodiments are as follows:

A VH domain, VL domain, set of HCDRs, set of LCDRs, or set of CDRs of:1126F1 (VH SEQ ID NO: 102; VL SEQ ID NO: 107), 1126G5 (VH SEQ ID NO:112; VL SEQ ID NO: 117), 1126H5 (VH SEQ ID NO: 122; VL SEQ ID NO: 127),1127D9 (VH SEQ ID NO: 132; VL SEQ ID NO: 137), 1127F9 (VH SEQ ID NO:142; VL SEQ ID NO: 147), 1131D7 (VH SEQ ID NO: 152; VL SEQ ID NO: 157),1131H2 (VH SEQ ID NO: 162; VL SEQ ID NO: 167), 1132A9 (VH SEQ ID NO:172; VL SEQ ID NO: 177), 1132H9 (VH SEQ ID NO: 182; VL SEQ ID NO: 187),1133C11 (VH SEQ ID NO: 192; VL SEQ ID NO: 197), 1134D9 (VH SEQ ID NO:202; VL SEQ ID NO: 207), 1145D1 (VH SEQ ID NO: 212; VL SEQ ID NO: 217),1146D7 (VH SEQ ID NO: 222; VL SEQ ID NO: 227), 1147D2 (VH SEQ ID NO:232; VL SEQ ID NO: 237), 1147G9 (VH SEQ ID NO: 242; VL SEQ ID NO: 247),1150F1 (VH SEQ ID NO: 252; VL SEQ ID NO: 257), 1152H5 (VH SEQ ID NO:262; VL SEQ ID NO: 267), 1155H1 (VH SEQ ID NO: 272; VL SEQ ID NO: 277),1158A1 (VH SEQ ID NO: 282; VL SEQ ID NO: 287), 1160E3 (VH SEQ ID NO:292; VL SEQ ID NO: 297), 1165D4 (VH SEQ ID NO: 302; VL SEQ ID NO: 307),1175H8 (VH SEQ ID NO: 312; VL SEQ ID NO: 317), 1211G10 (VH SEQ ID NO:322; VL SEQ ID NO: 327), 1214A1 (VH SEQ ID NO: 332; VL SEQ ID NO: 337),1214D10 (VH SEQ ID NO: 342; VL SEQ ID NO: 347), 1218H5 (VH SEQ ID NO:352; VL SEQ ID NO: 357), and 1230H7 (VH SEQ ID NO: 362; VL SEQ ID NO:367).

Still further preferred are a VH domain, VL domain, set of HCDRs, set ofLCDRs, or set of CDRs of 1083H4 (VH SEQ ID NO: 22; VL SEQ ID NO: 27),1227H8 (VH SEQ ID NO: 372; VL SEQ ID NO: 377) and 1230D8 (VH SEQ ID NO:382; VL SEQ ID NO: 387).

In a highly preferred embodiment, a VH domain is provided with the aminoacid sequence of SEQ ID NO: 192, this being termed “1133C11 VH domain”.In a further highly preferred embodiment, a VL domain is provided withthe amino acid sequence of SEQ ID NO: 197, this being termed “1133C11 VLdomain”. A highly preferred antibody antigen-binding site provided inaccordance with the present invention is composed of the 1133C11 VHdomain, SEQ ID NO: 192, and the 1133C11 VL domain, SEQ ID NO: 197. Thisantibody antigen-binding site may be provided within any desiredantibody molecule format, e.g. scFv, Fab, IgG, IgG4 etc., as isdiscussed further elsewhere herein.

In a further highly preferred embodiment, a VH domain is provided withthe amino acid sequence of SEQ ID NO: 392, this being termed “1252A5 VHdomain”. In a further highly preferred embodiment, a VL domain isprovided with the amino acid sequence of SEQ ID NO: 397, this beingtermed “1252A5 VL domain”. A highly preferred antibody antigen-bindingsite provided in accordance with the present invention is composed ofthe 1252A5 VH domain, SEQ ID NO: 392, and the 1252A5 VL domain, SEQ IDNO: 397. This antibody antigen-binding site may be provided within anydesired antibody molecule format, e.g. scFv, Fab, IgG, IgG4 etc., as isdiscussed further elsewhere herein.

In a further highly preferred embodiment, the present invention providesan IgG4 antibody molecule comprising the 1252A5 VH domain, SEQ ID NO:392, and the 1252A5 VL domain, SEQ ID NO: 397. This is termed herein“1252A5 IgG4”.

Other IgG or other antibody molecules comprising the 1252A5 VH domain,SEQ ID NO: 392, and/or the 1252A5 VL domain, SEQ ID NO: 397, areprovided by the present invention, as are other antibody moleculescomprising the 1252A5 set of HCDRs (SEQ ID NOS: 393, 394 and 395) withinan antibody VH domain, and/or the 1252A5 set of LCDRs (SEQ ID NOS: 398,399 and 400) within an antibody VL domain. As noted, the presentinvention provides a specific binding member which binds human NGF andwhich comprises the 1252A5 VH domain (SEQ ID NO: 392) and/or the 1252A5VL domain (SEQ ID NO: 397). Properties of such a specific binding memberare disclosed herein.

Generally, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below a VH domainalone may be used to bind antigen. In one preferred embodiment, the1252A5 VH domain (SEQ ID NO: 392) is paired with the 1252A5 VL domain(SEQ ID NO: 397), so that an antibody antigen-binding site is formedcomprising both the 1252A5 VH and VL domains. Analogous embodiments areprovided for the other VH and VL domains disclosed herein. In otherembodiments, the 1252A5 VH is paired with a VL domain other than the1252A5 VL. Light-chain promiscuity is well established in the art.Again, analogous embodiments are provided by the invention for the otherVH and VL domains disclosed herein.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in specific binding members for NGF can beobtained by means of methods of sequence alteration or mutation andscreening. Such methods are also provided by the present invention.

In accordance with further aspects of the present invention there isprovided a specific binding member which competes for binding to antigenwith any specific binding member which both binds the antigen andcomprises a specific binding member, VH and/or VL domain disclosedherein, or HCDR3 disclosed herein, or variant of any of these.Competition between binding members may be assayed easily in vitro, forexample using ELISA and/or by tagging a specific reporter molecule toone binding member which can be detected in the presence of one or moreother untagged binding members, to enable identification of specificbinding members which bind the same epitope or an overlapping epitope.

Thus, a further aspect of the present invention provides a specificbinding member comprising a human antibody antigen-binding site thatcompetes with an antibody molecule, for example especially 1252A5 orother preferred scFv and/or IgG4, for binding to NGF. In further aspectsthe present invention provides a specific binding member comprising ahuman antibody antigen-binding site which competes with an antibodyantigen-binding site for binding to NGF, wherein the antibodyantigen-binding site is composed of a VH domain and a VL domain, andwherein the VH and VL domains comprise a set of CDRs of the 1133C11,1021E5, 1252A5 or other lineage, disclosed herein.

Various methods are available in the art for obtaining antibodiesagainst NGF and which may compete with a 1252A5 or other antibodymolecule, an antibody molecule with a 1252A5 or other set of CDRs, or anantibody molecule with a set of CDRs of 1252A5 or other lineage, forbinding to NGF.

In a further aspect, the present invention provides a method ofobtaining one or more specific binding members able to bind the antigen,the method including bringing into contact a library of specific bindingmembers according to the invention and said antigen, and selecting oneor more specific binding members of the library able to bind saidantigen.

The library may be displayed on particles or molecular complexes, e.g.replicable genetic packages such as yeast, bacterial or bacteriophage(e.g. T7) particles, or covalent, ribosomal or other in vitro displaysystems, each particle or molecular complex containing nucleic acidencoding the antibody VH variable domain displayed on it, and optionallyalso a displayed VL domain if present.

Following selection of specific binding members able to bind the antigenand displayed on bacteriophage or other library particles or molecularcomplexes, nucleic acid may be taken from a bacteriophage or otherparticle or molecular complex displaying a said selected specificbinding member. Such nucleic acid may be used in subsequent productionof a specific binding member or an antibody VH or VL variable domain byexpression from nucleic acid with the sequence of nucleic acid takenfrom a bacteriophage or other particle or molecular complex displaying asaid selected specific binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected specific binding membermay be provided in isolated form, as may a specific binding membercomprising such a VH domain.

Ability to bind NGF may be further tested, also ability to compete withe.g. 1252A5 (e.g. in scFv format and/or IgG format, e.g. IgG4) forbinding to NGF. Ability to neutralise NGF may be tested, as discussedfurther below.

A specific binding member according to the present invention may bindNGF with the affinity of a 1252A5 or other antibody molecule, e.g. scFv,or preferably 1252A5 or other IgG4, or with an affinity that is better.

A specific binding member according to the present invention mayneutralise NGF with the potency of a 1252A5 or other antibody molecule,e.g. scFv, or preferably 1252A5 or other IgG4, or with a potency that isbetter.

Binding affinity and neutralisation potency of different specificbinding members can be compared under appropriate conditions.

The antibodies of the present invention have a number of advantages overexisting commercially available anti-NGF antibodies. For example, thepresent invention provides human or germlined antibodies, which areexpected to display a lower degree of immunogenicity when chronically orrepeatedly administered to humans for therapeutic or diagnostic use.Further, the present invention provides antibodies that are more potentneutralisers of NGF and therefore a desired therapeutic or diagnosticeffect may be achieved using less antibody material. In addition, in oneembodiment of the invention, the potency for inhibition of the NGF/TrKAreceptor interaction is greater than that observed for inhibition of theNGF/p75 receptor interaction. This may confer advantages over otherapparently non-selective NGF antagonist treatments in this regard,either in the magnitude or nature of the therapeutic effect achieved, orin reducing undesirable side effects.

The invention also provides heterogeneous preparations comprisinganti-NGF antibody molecules. For example, such preparations may bemixtures of antibodies with full-length heavy chains and heavy chainslacking the C-terminal lysine, with various degrees of glycosylationand/or with derivatized amino acids, such as cyclization of anN-terminal glutamic acid to form a pyroglutamic acid residue.

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a specific binding member, VH domainand/or VL domains according to the present invention, and methods ofpreparing a specific binding member, a VH domain and/or a VL domain ofthe invention, which comprise expressing said nucleic acid underconditions to bring about production of said specific binding member, VHdomain and/or VL domain, and recovering it.

A further aspect of the present invention provides nucleic acid,generally isolated, encoding an antibody VH variable domain and/or VLvariable domain disclosed herein.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein,especially a VH CDR selected from: 1133C11 (VH CDR1 SEQ ID NO: 193, VHCDR2 SEQ ID NO: 194, and VH CDR3 SEQ ID NO: 195), 1152H5 (VH CDR1 SEQ IDNO: 263, VH CDR2 SEQ ID NO: 264, and VH CDR3 SEQ ID NO: 265), and 1252A5(VH CDR1 SEQ ID NO: 393, VH CDR2 SEQ ID NO: 394, and VH CDR3 SEQ ID NO:395), or a VL CDR selected from: 1133C11 (VL CDR1 SEQ ID NO: 198, VLCDR2 SEQ ID NO: 199, and VL CDR3 SEQ ID NO: 200), 1152H5 (VL CDR1 SEQ IDNO: 268, VL CDR2 SEQ ID NO: 269, and VL CDR3 SEQ ID NO: 270), and 1252A5(VL CDR1 SEQ ID NO: 398, VL CDR2 SEQ ID NO: 399, and VL CDR3 SEQ ID NO:400), most preferably 1252A5 VH CDR3 (SEQ ID NO: 395). Nucleic acidencoding the 1252A5 set of CDRs, nucleic acid encoding the 1252A5 set ofHCDRs and nucleic acid encoding the 1252A5 set of LCDRs are alsoprovided by the present invention, as are nucleic acids encodingindividual CDRs, HCDRs, LCDs and sets of CDRs, HCDRs, LCDRs of the1252A5, 1133C11 or 1021E5 lineage.

A further aspect provides a host cell transformed with nucleic acid ofthe invention.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and specificbinding members comprising a VH and/or VL domain are provided as furtheraspects of the present invention.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Further aspects of the present invention provide for compositionscontaining specific binding members of the invention, and their use inmethods of inhibiting or neutralising NGF, including methods oftreatment of the human or animal body by therapy.

Specific binding members according to the invention may be used in amethod of treatment or diagnosis of the human or animal body, such as amethod of treatment (which may include prophylactic treatment) of adisease or disorder in a human patient which comprises administering tosaid patient an effective amount of a specific binding member of theinvention. Conditions treatable in accordance with the present inventioninclude any in which NGF plays a role, especially pain, asthma, chronicobstructive pulmonary disease, pulmonary fibrosis, other diseases ofairway inflammation, diabetic neuropathy, HIV, cardiac arrhythmias,arthritis, psoriasis and cancer.

These and other aspects of the invention are described in further detailbelow.

Terminology

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B″ is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

NGF

NGF (also known as beta-NGF) is nerve growth factor. In the context ofthe present invention, NGF is normally human NGF, although it may benon-human NGF (e.g. non-human primate NGF and/or rat NGF and/or mouseNGF). NGF is also referred to in places as “the antigen”.

NGF used in an assay described herein is normally human, rat or mouseNGF, but NGF from another non-human animal could be used, e.g. non-humanprimate NGF.

Pain

This describes, as is well known in the art, sensation of pain, and mayencompass one or more, or all, of the following:

-   -   hyperalgesia (exaggerated pain response to a normally painful        stimulus);    -   allodynia (sensation of pain caused by a stimulus that is not        normally painful);    -   spontaneous sensation of pain caused by any mechanism in the        absence of any apparent external influence;    -   pain evoked by physical stimuli, such as heat, warmth, cold,        pressure, vibration, static or dynamic touch, or body posture        and movement;    -   somatic and visceral pain caused by any mechanism, for example,        trauma, infection, inflammation, metabolic disease, stroke or        neurological disease.

Pain may for example be acute pain, short-term pain, persistentnociceptive pain, or persistent or chronic neuropathic pain.

Specific Binding Member

This describes a member of a pair of molecules that have bindingspecificity for one another. The members of a specific binding pair maybe naturally derived or wholly or partially synthetically produced. Onemember of the pair of molecules has an area on its surface, or a cavity,which specifically binds to and is therefore complementary to aparticular spatial and polar organisation of the other member of thepair of molecules. Thus the members of the pair have the property ofbinding specifically to each other. Examples of types of specificbinding pairs are antigen-antibody, biotin-avidin, hormone-hormonereceptor, receptor-ligand, enzyme-substrate. The present invention isconcerned with antigen-antibody type reactions.

A specific binding member normally comprises a molecule having anantigen-binding site. For example, a specific binding member may be anantibody molecule or a non-antibody protein that comprises anantigen-binding site. An antigen binding site may be provided by meansof arrangement of CDRs on non-antibody protein scaffolds such asfibronectin or cytochrome B etc. (Haan & Maggos, 2004; Koide et al.,1998; Nygren et al., 1997), or by randomising or mutating amino acidresidues of a loop within a protein scaffold to confer bindingspecificity for a desired target. Scaffolds for engineering novelbinding sites in proteins have been reviewed in detail by Nygren et al.(1997). Protein scaffolds for antibody mimics are disclosed inWO/0034784 in which the inventors describe proteins (antibody mimics)that include a fibronectin type III domain having at least onerandomised loop. A suitable scaffold into which to graft one or moreCDRs, e.g. a set of HCDRs, may be provided by any domain member of theimmunoglobulin gene superfamily. The scaffold may be a human ornon-human protein.

An advantage of a non-antibody protein scaffold is that it may providean antigen-binding site in a scaffold molecule that is smaller and/oreasier to manufacture than at least some antibody molecules. Small sizeof a specific binding member may confer useful physiological propertiessuch as an ability to enter cells, penetrate deep into tissues or reachtargets within other structures, or to bind within protein cavities ofthe target antigen.

Use of antigen binding sites in non-antibody protein scaffolds isreviewed in Wess, 2004. Typical are proteins having a stable backboneand one or more variable loops, in which the amino acid sequence of theloop or loops is specifically or randomly mutated to create anantigen-binding site having specificity for binding the target antigen.Such proteins include the IgG-binding domains of protein A from S.aureus, transferrin, tetranectin, fibronectin (e.g. 10th fibronectintype III domain) and lipocalins. Other approaches include synthetic“Microbodies” (Selecore GmbH), which are based on cyclotides—smallproteins having intra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, aspecific binding member according to the present invention may compriseother amino acids, e.g. forming a peptide or polypeptide, such as afolded domain, or to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. Specific bindingmembers of the invention may carry a detectable label, or may beconjugated to a toxin or a targeting moiety or enzyme (e.g. via apeptidyl bond or linker). For example, a specific binding member maycomprise a catalytic site (e.g. in an enzyme domain) as well as anantigen binding site, wherein the antigen binding site binds to theantigen and thus targets the catalytic site to the antigen. Thecatalytic site may inhibit biological function of the antigen, e.g. bycleavage.

Although, as noted, CDRs can be carried by scaffolds such as fibronectinor cytochrome B (Haan & Maggos, 2004; Koide et al., 1998; Nygren et al.,1997), the structure for carrying a CDR or a set of CDRs of theinvention will generally be of an antibody heavy or light chain sequenceor substantial portion thereof in which the CDR or set of CDRs islocated at a location corresponding to the CDR or set of CDRs ofnaturally occurring VH and VL antibody variable domains encoded byrearranged immunoglobulin genes. The structures and locations ofimmunoglobulin variable domains may be determined by reference to(Kabat, et al., 1987, and updates thereof, now available on the Internet(http://immuno.bme.nwu.edu or find “Kabat” using any search engine).

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antibody antigen-binding site. Antibody fragments thatcomprise an antibody antigen-binding site are molecules such as Fab,scFv, Fv, dAb, Fd; and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that retain the specificity of the original antibody.Such techniques may involve introducing DNA encoding the immunoglobulinvariable region, or the CDRs, of an antibody to the constant regions, orconstant regions plus framework regions, of a different immunoglobulin.See, for instance, EP-A-184187, GB 2188638A or EP-A-239400, and a largebody of subsequent literature. A hybridoma or other cell producing anantibody may be subject to genetic mutation or other changes, which mayor may not alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any specific binding member orsubstance having an antibody antigen-binding site with the requiredspecificity. Thus, this term covers antibody fragments and derivatives,including any polypeptide comprising an antibody antigen-binding site,whether natural or wholly or partially synthetic. Chimeric moleculescomprising an antibody antigen-binding site, or equivalent, fused toanother polypeptide are therefore included. Cloning and expression ofchimeric antibodies are described in EP-A-0120694 and EP-A-0125023, anda large body of subsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann & Dubel (2001).Phage display, another established technique for generating specificbinding members has been described in detail in many publications suchas Kontermann & Dubel (2001) and WO92/01047 (discussed further below).Transgenic mice in which the mouse antibody genes are inactivated andfunctionally replaced with human antibody genes while leaving intactother components of the mouse immune system, can be used for isolatinghuman antibodies (Mendez et al., 1997).

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.(2000) or Krebs et al. (2001).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003), whichconsists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments (vii)single chain Fv molecules (scFv), wherein a VH domain and a VL domainare linked by a peptide linker which allows the two domains to associateto form an antigen binding site (Bird et al., 1988; Huston et al.,1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and(ix) “diabodies”, multivalent or multispecific fragments constructed bygene fusion (WO94/13804; Holliger et al., 1993). Fv, scFv or diabodymolecules may be stabilised by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996). Minibodiescomprising a scFv joined to a CH3 domain may also be made (Hu et al.,1996).

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain (Holt et al., 2003). VH dAbs occur naturally in camelids (e.g.camel, llama) and may be produced by immunising a camelid with a targetantigen, isolating antigen-specific B cells and directly cloning dAbgenes from individual B cells. dAbs are also producible in cell culture.Their small size, good solubility and temperature stability makes themparticularly physiologically useful and suitable for selection andaffinity maturation. A specific binding member of the present inventionmay be a dAb comprising a VH or VL domain substantially as set outherein, or a VH or VL domain comprising a set of CDRs substantially asset out herein.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger & Winter, 1993), e.g. prepared chemically or from hybridhybridomas, or may be any of the bispecific antibody fragments mentionedabove. Examples of bispecific antibodies include those of the BiTE™technology in which the binding domains of two antibodies with differentspecificity can be used and directly linked via short flexible peptides.This combines two antibodies on a short single polypeptide chain.Diabodies and scFv can be constructed without an Fc region, using onlyvariable domains, potentially reducing the effects of anti-idiotypicreaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against NGF, then a library can be made where the other arm isvaried and an antibody of appropriate specificity selected. Bispecificwhole antibodies may be made by knobs-into-holes engineering (Ridgewayet al., 1996).

Antigen-Binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that specifically binds to and is complementary to allor part of the target antigen. Where an antigen is large, an antibodymay only bind to a particular part of the antigen, which part is termedan epitope. An antibody antigen-binding site may be provided by one ormore antibody variable domains. Preferably, an antibody antigen-bindingsite comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

Specific

This may be used to refer to the situation in which one member of aspecific binding pair will not show any significant binding to moleculesother than its specific binding partner(s). The term is also applicablewhere e.g. an antigen-binding site is specific for a particular epitopethat is carried by a number of antigens, in which case the specificbinding member carrying the antigen-binding site will be able to bind tothe various antigens carrying the epitope.

Isolated

This refers to the state in which specific binding members of theinvention, or nucleic acid encoding such binding members, will generallybe in accordance with the present invention. Isolated members andisolated nucleic acid will be free or substantially free of materialwith which they are naturally associated such as other polypeptides ornucleic acids with which they are found in their natural environment, orthe environment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo. Members and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themembers will normally be mixed with gelatin or other carriers if used tocoat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Specific binding members may be glycosylated, eithernaturally or by systems of heterologous eukaryotic cells (e.g. CHO orNS0 (ECACC 85110503) cells, or they may be (for example if produced byexpression in a prokaryotic cell) unglycosylated.

DETAILED DESCRIPTION

As noted above, a specific binding member in accordance with the presentinvention preferably neutralises NGF. The degree to which an antibodyneutralises NGF is referred to as its neutralising potency.

Potency is normally expressed as an IC50 value, in nM unless otherwisestated. IC50 is the median inhibitory concentration of an antibodymolecule. In functional assays, IC50 is the concentration that reduces abiological response by 50% of its maximum. In ligand-binding studies,IC50 is the concentration that reduces receptor binding by 50% ofmaximal specific binding level.

IC50 may be calculated by plotting % biological response (representede.g. by calcium ion mobilisation in a FLIPR assay, by survival in a PC12assay, or by proliferation in a TF-1 proliferation assay) or % specificreceptor binding as a function of the log of the specific binding memberconcentration, and using a software program such as Prism (GraphPad) tofit a sigmoidal function to the data to generate IC50 values, forexample as described in Example 2, 5, 6 or 9 herein.

A specific binding member in accordance with the present inventionpreferably inhibits human NGF-evoked intracellular calcium mobilisationin cells expressing TrkA receptor, e.g. cells recombinantly transfectedwith a TrkA gene, for instance HEK cells. In a “FLIPR” calciummobilisation assay as described in Example 2 herein, a specific bindingmember according to the invention preferably has a potency (IC50) forneutralising human NGF of or less than 600, 100, 90, 80, 70, 60, 50, 40,30, 20 or 10 nM. Normally, a specific binding member of the inventionhas a potency of 5 nM or less, preferably 2.5 nM or less, morepreferably 1 nM or less. In particularly preferred embodiments, thepotency is 0.5 nM or less, e.g. 0.4 nM or less; 0.3 nM or less; 0.2 nMor less; or 0.15 nM or less. In some embodiments, the potency may beabout 0.1 nM.

Potency may be between 0.1-100 nM, 0.1-50 nM, 0.1-10 nM, or 0.1-1.0 nM.For example, potency may be 0.1-5.0 nM, 0.2-5.0 nM, 0.3-5.0 nM, or0.3-0.4 nM.

In some embodiments of the invention, the neutralising potency of a nonpotency-optimised specific binding member in a HEK cell assay asdescribed herein is about 1.8 to 560 nM for human NGF and/or about 2.9to 620 nM for rat NGF. In some embodiments, the neutralizing potency ofpotency-optimised binding members in HEK cell assays as described hereinare about 0.12 to 120 nM for human NGF, about 0.11 to 37 nM for rat NGFand about 0.11 to 71 nM for mouse NGF. However, these are examples onlyand higher potencies may be achieved. Although potency optimisation maybe used to generate higher potency specific binding members from a givenspecific binding member, it is also noted that high potency specificbinding members may be obtained even without potency optimisation.

A specific binding member in accordance with the present inventionpreferably inhibits NGF-maintained serum-deprived PC12 cell survival.The neutralising potency of a specific binding member of the presentinvention in a PC12 survival assay for human NGF as described herein inExample 5 is generally 1500 nM or less, and is preferably 50 nM or less,or 10 nM or less. As explained above and as demonstrated herein,potency-optimisation may be used to achieve higher anti-NGF potencies.Preferably, a specific binding member has a potency of or less than 5nM, 4 nM, 3 nM, 2 nM, 1.5 nM, 1 nM or 0.5 nM. In some embodiments,potency is about 0.1 nM or more, 0.2 nM or more. Thus, potency may bebetween 0.1 or 0.2 nM and 0.5, 1.5, 5 or 50 nM.

In some embodiments of the invention, the neutralizing potency of apotency optimised specific binding member in a PC12 survival assay asdescribed herein is about 0.2 to 670 nM for human NGF and is about 0.2to 54 nM for rat NGF.

A specific binding member in accordance with the present inventionpreferably inhibits NGF-stimulated TF-1 cell proliferation. Theneutralising potency of a specific binding member (normally apotency-optimised specific binding member) of the present invention in aTF-1 proliferation assay for human NGF as described herein in Example 6is generally 5 nM or less, preferably 1 nM or less. Preferably, aspecific binding member of the invention has a potency of or less than0.7, 0.6, 0.5, 0.45, 0.4, 0.3, 0.2 or 0.1 nM for human NGF. For example,potency may be between 0.05-0.1 nm, 0.05-0.2 nM, 0.05-0.3 nM, 0.05-0.4nM, or 0.05-0.5 nM.

In some embodiments of the invention, the neutralizing potency of apotency optimised specific binding member in a TF-1 proliferation assayas described herein is about 0.08 to 0.7 nM for human NGF, about 0.07 to1.9 nM for rat NGF and about 0.07 to 1.4 nM for mouse NGF.

A specific binding member in accordance with the present inventionpreferably inhibits NGF binding to a TrkA and/or p75 receptor,preferably a human TrkA and/or p75 receptor. The invention also extendsmore generally to a specific binding member that preferentially blocksNGF binding to TrkA receptor over NGF binding to p75 receptor. Theneutralising potency of a specific binding member (normally apotency-optimised specific binding member) of the present invention in aTrkA receptor binding assay as described herein in Example 9 isgenerally 2.5 nM or less, preferably 1 nM or less for neutralising humanNGF. Preferably, a specific binding member of the invention has apotency of or less than 0.5, 0.4, 0.3, 0.2, 0.1 or 0.075 nM forneutralising human NGF binding to TrkA. For example, potency may bebetween 0.05-0.1 nm, 0.05-0.2 nM, 0.05-0.3 nM, 0.05-0.4 nM, or 0.05-0.5nM.

The neutralising potency of a specific binding member (normally apotency-optimised specific binding member) of the present invention in ap75 receptor binding assay as described herein in Example 9 is generally1.5 nM or less, preferably 1 nM or less for neutralising human NGF.Preferably, a specific binding member of the invention has a potency ofor less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 nM forneutralising human NGF binding to p75. For example, potency may bebetween 0.1-0.2 nM, 0.1-0.3 nM, 0.1-0.4 nM, 0.1-0.5 nM, or 0.1-0.6 nM.

Some preferred specific binding members according to the presentinvention inhibit NGF (e.g. human and/or rat NGF) binding to TrkAreceptor preferentially over NGF binding to p75 receptor. Accordingly,in some embodiments a specific binding member of the invention has alower binding inhibition constant, Ki, for inhibition of NGF (e.g. humanand/or rat NGF) binding to TrkA than for NGF binding to p75. Ki may becalculated using the formula set out in Example 9. Alternatively,binding inhibition constants can be expressed as pKi, which can becalculated as −log₁₀Ki. Thus, a specific binding member of the inventionpreferably has a higher pKi value for inhibition of NGF binding to TrkAthan to p75.

Preferably, a specific binding member according to the invention bindshuman NGF and/or rat NGF with an affinity of or less than 1, 0.8, 0.7,0.6, 0.5, 0.4, 0.3 or 0.2 nM. For example, a specific binding member maybind human NGF with an affinity of about 0.25-0.44 nM and rat NGF withan affinity of about 0.25-0.70 nM.

As noted above, variants of antibody molecules disclosed herein may beproduced and used in the present invention. Following the lead ofcomputational chemistry in applying multivariate data analysistechniques to the structure/property-activity relationships (Wold, etal. 1984) quantitative activity-property relationships of antibodies canbe derived using well-known mathematical techniques such as statisticalregression, pattern recognition and classification (Norman et al. 1998;Kandel & Backer, 1995; Krzanowski, 2000; Witten & Frank, 1999; Denison(Ed), 2002; Ghose & Viswanadhan). The properties of antibodies can bederived from empirical and theoretical models (for example, analysis oflikely contact residues or calculated physicochemical property) ofantibody sequence, functional and three-dimensional structures and theseproperties can be considered singly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis formed by six loops of polypeptide: three from the light chainvariable domain (VL) and three from the heavy chain variable domain(VH). Analysis of antibodies of known atomic structure has elucidatedrelationships between the sequence and three-dimensional structure ofantibody combining sites (Chothia et al. 1992; Al-Lazikani, et al.1997). These relationships imply that, except for the third region(loop) in VH domains, binding site loops have one of a small number ofmain-chain conformations: canonical structures. The canonical structureformed in a particular loop has been shown to be determined by its sizeand the presence of certain residues at key sites in both the loop andin framework regions (Chothia et al. and Al-Lazikani et al., supra).

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimization experiments. In astructural approach, a model can be created of the antibody molecule(Chothia, et al. 1986) using any freely available or commercial packagesuch as WAM (Whitelegg & Rees, 2000). A protein visualisation andanalysis software package such as Insight II (Accelerys, Inc.) or DeepView (Guex & Peitsch, 1997) may then be used to evaluate possiblesubstitutions at each position in the CDR. This information may then beused to make substitutions likely to have a minimal or beneficial effecton activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and specific bindingmembers generally are available in the art. Variant sequences may bemade, with substitutions that may or may not be predicted to have aminimal or beneficial effect on activity, and tested for ability to bindand/or neutralise NGF and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, maybe 5, 4, 3, 2 or 1. Alterations may be made in one ormore framework regions and/or one or more CDRs.

Preferably alterations do not result in loss of function, so a specificbinding member comprising a thus-altered amino acid sequence preferablyretains an ability to bind and/or neutralise NGF. More preferably, itretains the same quantitative binding and/or neutralising ability as aspecific binding member in which the alteration is not made, e.g. asmeasured in an assay described herein. Most preferably, the specificbinding member comprising a thus-altered amino acid sequence has animproved ability to bind or neutralise NGF.

Alteration may comprise replacing one or more amino acid residue with anon-naturally occurring or non-standard amino acid, modifying one ormore amino acid residue into a non-naturally occurring or non-standardform, or inserting one or more non-naturally occurring or non-standardamino acid into the sequence. Preferred numbers and locations ofalterations in sequences of the invention are described elsewhereherein. Naturally occurring amino acids include the 20 “standard”L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C,K, R, H, D, E by their standard single-letter codes. Non-standard aminoacids include any other residue that may be incorporated into apolypeptide backbone or result from modification of an existing aminoacid residue. Non-standard amino acids may be naturally occurring ornon-naturally occurring. Several naturally occurring non-standard aminoacids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine,3-methylhistidine, N-acetylserine, etc. (Voet & Voet, 1995). Those aminoacid residues that are derivatised at their N-alpha position will onlybe located at the N-terminus of an amino-acid sequence. Normally in thepresent invention an amino acid is an L-amino acid, but in someembodiments it may be a D-amino acid. Alteration may therefore comprisemodifying an L-amino acid into, or replacing it with, a D-amino acid.Methylated, acetylated and/or phosphorylated forms of amino acids arealso known, and amino acids in the present invention may be subject tosuch modification.

Amino acid sequences in antibody domains and specific binding members ofthe invention may comprise non-natural or non-standard amino acidsdescribed above. In some embodiments non-standard amino acids (e.g.D-amino acids) may be incorporated into an amino acid sequence duringsynthesis, while in other embodiments the non-standard amino acids maybe introduced by modification or replacement of the “original” standardamino acids after synthesis of the amino acid sequence.

Use of non-standard and/or non-naturally occurring amino acids increasesstructural and functional diversity, and can thus increase the potentialfor achieving desired NGF binding and neutralising properties in aspecific binding member of the invention. Additionally, D-amino acidsand analogues have been shown to have better pharmacokinetic profilescompared with standard L-amino acids, owing to in vivo degradation ofpolypeptides having L-amino acids after administration to an animal.

As noted above, a CDR amino acid sequence substantially as set outherein is preferably carried as a CDR in a human antibody variabledomain or a substantial portion thereof. The HCDR3 sequencessubstantially as set out herein represent preferred embodiments of thepresent invention and it is preferred that each of these is carried as aHCDR3 in a human heavy chain variable domain or a substantial portionthereof.

Variable domains employed in the invention may be obtained or derivedfrom any germ-line or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A CDR sequence of the invention (e.g.CDR3) may be introduced into a repertoire of variable domains lacking aCDR (e.g. CDR3), using recombinant DNA technology.

For example, Marks et al. (1992) describe methods of producingrepertoires of antibody variable domains in which consensus primersdirected at or adjacent to the 5′ end of the variable domain area areused in conjunction with consensus primers to the third framework regionof human VH genes to provide a repertoire of VH variable domains lackinga CDR3. Marks et al. further describe how this repertoire may becombined with a CDR3 of a particular antibody. Using analogoustechniques, the CDR3-derived sequences of the present invention may beshuffled with repertoires of VH or VL domains lacking a CDR3, and theshuffled complete VH or VL domains combined with a cognate VL or VHdomain to provide specific binding members of the invention. Therepertoire may then be displayed in a suitable host system such as thephage display system of WO92/01047 or any of a subsequent large body ofliterature, including Kay, Winter & McCafferty (1996), so that suitablespecific binding members may be selected. A repertoire may consist offrom anything from 10⁴ individual members upwards, for example from 10⁶to 10⁸ or 10¹⁰ embers. Other suitable host systems include yeastdisplay, bacterial display, T7 display, ribosome display and covalentdisplay.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (1994), who describes the technique in relation to a β-lactamasegene but observes that the approach may be used for the generation ofantibodies.

A further alternative is to generate novel VH or VL regions carryingCDR-derived sequences of the invention using random mutagenesis of oneor more selected VH and/or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al.(1992), who used error-prone PCR. In preferred embodiments one or twoamino acid substitutions are made within a set of HCDRs and/or LCDRs.

Another method that may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al. (1994)and Schier et al. (1996).

All the above-described techniques are known as such in the art and theskilled person will be able to use such techniques to provide specificbinding members of the invention using routine methodology in the art.

A further aspect of the invention provides a method for obtaining anantibody antigen-binding site specific for NGF antigen, the methodcomprising providing by way of addition, deletion, substitution orinsertion of one or more amino acids in the amino acid sequence of a VHdomain set out herein a VH domain which is an amino acid sequencevariant of the VH domain, optionally combining the VH domain thusprovided with one or more VL domains, and testing the VH domain or VH/VLcombination or combinations to identify a specific binding member or anantibody antigen-binding site specific for NGF antigen and optionallywith one or more preferred properties, preferably ability to neutraliseNGF activity. Said VL domain may have an amino acid sequence which issubstantially as set out herein.

An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

In a preferred embodiment, 1252A5 VH domain (SEQ ID NO: 392) may besubject to mutation to provide one or more VH domain amino acid sequencevariants, optionally combined with 1252A5 VL (SEQ ID NO: 397).

A further aspect of the invention provides a method of preparing aspecific binding member specific for NGF antigen, which methodcomprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain which either include a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a VH        CDR3 such that said donor nucleic acid is inserted into the CDR3        region in the repertoire, so as to provide a product repertoire        of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting a specific binding member specific for NGF; and    -   (e) recovering said specific binding member or nucleic acid        encoding it.

Again, an analogous method may be employed in which a VL CDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain that either include a CDR3 to be replaced or lack a CDR3 encodingregion.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a specificbinding member or specific binding members specific for NGF.

In a preferred embodiment, one or more of 1252A5 HCDR1 (SEQ ID NO: 393),HCDR2 (SEQ ID NO: 394) and HCDR3 (SEQ ID NO: 395), or the 1252A5 set ofHCDRs, may be employed, and/or one or more of 1252A5 LCDR1 (SEQ ID NO:398), LCDR2 (SEQ ID NO: 399) and LCDR3 (SEQ ID NO: 400) or the 1252A5set of LCDRs may be employed.

In other analogous embodiments 1152H5, 1165D4 or 1230H7 is substitutedfor 1252A5.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRsand/or sets of LCDRs disclosed herein may be employed.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains of the invention to further protein sequences includingantibody constant regions, other variable domains (for example in theproduction of diabodies) or detectable/functional labels as discussed inmore detail elsewhere herein.

Although in a preferred aspect of the invention specific binding memberscomprising a pair of VH and VL domains are preferred, single bindingdomains based on either VH or VL domain sequences form further aspectsof the invention. It is known that single immunoglobulin domains,especially VH domains, are capable of binding target antigens in aspecific manner. For example, see the discussion of dAbs above.

In the case of either of the single specific binding domains, thesedomains may be used to screen for complementary domains capable offorming a two-domain specific binding member able to bind NGF.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inWO92/01047, in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain specific binding memberis selected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Markset al, ibid.

Specific binding members of the present invention may further compriseantibody constant regions or parts thereof, preferably human antibodyconstant regions or parts thereof. For example, a VL domain may beattached at its C-terminal end to antibody light chain constant domainsincluding human Cκ or Cλ chains, preferably Cλ chains. Similarly, aspecific binding member based on a VH domain may be attached at itsC-terminal end to all or part (e.g. a CH1 domain) of an immunoglobulinheavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE andIgM and any of the isotype sub-classes, particularly IgG1 and IgG4. IgG4is preferred. IgG4 is preferred because it does not bind complement anddoes not create effector functions. Any synthetic or other constantregion variant that has these properties and stabilizes variable regionsis also preferred for use in embodiments of the present invention.

Specific binding members of the invention may be labelled with adetectable or functional label. Detectable labels include radiolabelssuch as ¹³¹I or ⁹⁹Tc, which may be attached to antibodies of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include enzyme labels such as horseradishperoxidase. Labels further include chemical moieties such as biotin thatmay be detected via binding to a specific cognate detectable moiety,e.g. labelled avidin.

Specific binding members of the present invention are designed to beused in methods of diagnosis or treatment in human or animal subjects,preferably human.

Accordingly, further aspects of the invention provide methods oftreatment comprising administration of a specific binding member asprovided, pharmaceutical compositions comprising such a specific bindingmember, and use of such a specific binding member in the manufacture ofa medicament for administration, for example in a method of making amedicament or pharmaceutical composition comprising formulating thespecific binding member with a pharmaceutically acceptable excipient.

Clinical indications in which an anti-NGF antibody may be used toprovide therapeutic benefit include pain, asthma, chronic obstructivepulmonary disease, pulmonary fibrosis, other diseases of airwayinflammation, diabetic neuropathy, arthritis, psoriasis, cardiacarrhythmias, HIV and cancer. As already explained, anti-NGF treatment isindicated for all these diseases.

Anti-NGF treatment may be given orally, by injection (for example,subcutaneously, intravenously, intraperitoneal or intramuscularly), byinhalation, by the intravesicular route (instillation into the urinarybladder), or topically (for example intraocular, intranasal, rectal,into wounds, on skin). The route of administration can be determined bythe physicochemical characteristics of the treatment, by specialconsiderations for the disease or by the requirement to optimiseefficacy or to minimise side-effects.

It is envisaged that anti-NGF treatment will not be restricted to use inthe clinic. Therefore, subcutaneous injection using a needle free deviceis also preferred.

Combination treatments may be used to provide significant synergisticeffects, particularly the combination of an anti-NGF specific bindingmember with one or more other drugs. A specific binding member accordingto the present invention may be provided in combination or addition toshort or long acting analgesic, anti-inflammatory, anti-allergic,anti-asthmatic, anti-fibrotic, antiviral, chemotherapeutic agents andimmunotherapeutic agents.

Combination treatment with one or more short or long acting analgesicsand/or anti-inflammatory agents, such as opioids and non-steroidanti-inflammatory drugs (NSAIDs), may be employed for treatment ofconditions characterized by pain and/or inflammation for examplerheumatoid arthritis or post-surgical pain. Antibodies of the presentinvention can also be used in combination with anti-asthma,anti-allergic, or anti-fibrotic therapies, such as inhaled betaadrenoceptor agonists, steroids, cytokine antagonists, or other noveltherapeutic approaches for treatment of asthma, allergic asthma, otherallergic conditions, or any condition characterized by abnormalfibrosis. Antibodies of the present invention may also be used incombination with anti-infective agents, for example antiviral agents forthe treatment of HIV infection.

In accordance with the present invention, compositions provided may beadministered to individuals. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and may depend on the severity of the symptomsand/or progression of a disease being treated. Appropriate doses ofantibody are well known in the art; see Ledermann et al. (1991) andBagshawe (1991). Specific dosages indicated herein, or in thePhysician's Desk Reference (2003) as appropriate for the type ofmedicament being administered, may be used. A therapeutically effectiveamount or suitable dose of a specific binding member of the inventioncan be determined by comparing its in vitro activity and in vivoactivity in an animal model. Methods for extrapolation of effectivedosages in mice and other test animals to humans are known.

The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis or for treatment, the size and location ofthe area to be treated, the precise nature of the antibody (e.g. wholeantibody, fragment or diabody), and the nature of any detectable labelor other molecule attached to the antibody. A typical antibody dose willbe in the range 100 μg to 1 g for systemic applications, and 1 μg to 1mg for topical applications. Typically, the antibody will be a wholeantibody, preferably the IgG4 isotype. This is a dose for a singletreatment of an adult patient, which may be proportionally adjusted forchildren and infants, and also adjusted for other antibody formats inproportion to molecular weight. Treatments may be repeated at daily,twice-weekly, weekly or monthly intervals, at the discretion of thephysician. In preferred embodiments of the present invention, treatmentis periodic, and the period between administrations is about two weeksor more, preferably about three weeks or more, more preferably aboutfour weeks or more, or about once a month. In other preferredembodiments of the invention, treatment may be given before, and/orafter surgery, and more preferably, may be administered or applieddirectly at the anatomical site of surgical treatment.

Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder, liquid or semi-solid form. A tablet may comprise asolid carrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally comprise a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Specific binding members of the present invention may be formulated inliquid, semi-solid or solid forms depending on the physicochemicalproperties of the molecule and the route of delivery. Formulations mayinclude excipients, or combinations of excipients, for example: sugars,amino acids and surfactants. Liquid formulations may include a widerange of antibody concentrations and pH. Solid formulations may beproduced by lyophilisation, spray drying, or drying by supercriticalfluid technology, for example. Formulations of anti-NGF will depend uponthe intended route of delivery: for example, formulations for pulmonarydelivery may consist of particles with physical properties that ensurepenetration into the deep lung upon inhalation; topical formulations mayinclude viscosity modifying agents, which prolong the time that the drugis resident at the site of action. In certain embodiments, the specificbinding member may be prepared with a carrier that will protect thespecific binding member against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are known to thoseskilled in the art. See, e.g., Robinson, 1978.

The present invention provides a method comprising causing or allowingbinding of a specific binding member as provided herein to NGF. Asnoted, such binding may take place in vivo, e.g. followingadministration of a specific binding member, or nucleic acid encoding aspecific binding member, or it may take place in vitro, for example inELISA, Western blotting, immunocytochemistry, immuno-precipitation,affinity chromatography, or cell based assays such as a TF-1 assay.

The amount of binding of specific binding member to NGF may bedetermined. Quantitation may be related to the amount of the antigen ina test sample, which may be of diagnostic interest.

A kit comprising a specific binding member or antibody moleculeaccording to any aspect or embodiment of the present invention is alsoprovided as an aspect of the present invention. In a kit of theinvention, the specific binding member or antibody molecule may belabelled to allow its reactivity in a sample to be determined, e.g. asdescribed further below. Components of a kit are generally sterile andin sealed vials or other containers. Kits may be employed in diagnosticanalysis or other methods for which antibody molecules are useful. A kitmay contain instructions for use of the components in a method, e.g. amethod in accordance with the present invention. Ancillary materials toassist in or to enable performing such a method may be included within akit of the invention.

The reactivities of antibodies in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the antibody. Bound antigen is physicallyseparated from unbound antigen and the amount of radioactive antigenbound to the antibody determined. The more antigen there is in the testsample the less radioactive antigen will bind to the antibody. Acompetitive binding assay may also be used with non-radioactive antigen,using antigen or an analogue linked to a reporter molecule. The reportermolecule may be a fluorochrome, phosphor or laser dye with spectrallyisolated absorption or emission characteristics. Suitable fluorochromesinclude fluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes, which catalyse reactions that develop, or change colours orcause changes in electrical properties, for example. They may bemolecularly excitable, such that electronic transitions between energystates result in characteristic spectral absorptions or emissions. Theymay include chemical entities used in conjunction with biosensors.Biotin/avidin or biotin/streptavidin and alkaline phosphatase detectionsystems may be employed.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in samples (normal and test).

The present invention also provides the use of a specific binding memberas above for measuring antigen levels in a competition assay, that is tosay a method of measuring the level of antigen in a sample by employinga specific binding member as provided by the present invention in acompetition assay. This may be where the physical separation of boundfrom unbound antigen is not required. Linking a reporter molecule to thespecific binding member so that a physical or optical change occurs onbinding is one possibility. The reporter molecule may directly orindirectly generate detectable, and preferably measurable, signals. Thelinkage of reporter molecules may be directly or indirectly, covalently,e.g. via a peptide bond or non-covalently. Linkage via a peptide bondmay be as a result of recombinant expression of a gene fusion encodingantibody and reporter molecule.

The present invention also provides for measuring levels of antigendirectly, by employing a specific binding member according to theinvention for example in a biosensor system.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

As noted, in various aspects and embodiments, the present inventionextends to a specific binding member that competes for binding to NGFwith any specific binding member defined herein, e.g. 1252A5 IgG4.Competition between binding members may be assayed easily in vitro, forexample by tagging a specific reporter molecule to one binding memberwhich can be detected in the presence of other untagged bindingmember(s), to enable identification of specific binding members whichbind the same epitope or an overlapping epitope.

Competition may be determined for example using ELISA in which NGF isimmobilised to a plate and a first tagged binding member along with oneor more other untagged binding members is added to the plate. Presenceof an untagged binding member that competes with the tagged bindingmember is observed by a decrease in the signal emitted by the taggedbinding member.

In testing for competition a peptide fragment of the antigen may beemployed, especially a peptide including or consisting essentially of anepitope of interest. A peptide having the epitope sequence plus one ormore amino acids at either end may be used. Specific binding membersaccording to the present invention may be such that their binding forantigen is inhibited by a peptide with or including the sequence given.In testing for this, a peptide with either sequence plus one or moreamino acids may be used.

Specific binding members that bind a specific peptide may be isolatedfor example from a phage display library by panning with the peptide(s).

The present invention further provides an isolated nucleic acid encodinga specific binding member of the present invention. Nucleic acid mayinclude DNA and/or RNA. In a preferred aspect, the present inventionprovides a nucleic acid that codes for a CDR or set of CDRs or VH domainor VL domain or antibody antigen-binding site or antibody molecule, e.g.scFv or IgG4, of the invention as defined above.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell thatcomprises one or more constructs as above. A nucleic acid encoding anyCDR or set of CDRs or VH domain or VL domain or antibody antigen-bindingsite or antibody molecule, e.g. scFv or IgG4 as provided, itself formsan aspect of the present invention, as does a method of production ofthe encoded product, which method comprises expression from encodingnucleic acid therefor. Expression may conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression a VH or VL domain,or specific binding member may be isolated and/or purified using anysuitable technique, then used as appropriate.

Specific binding members, VH and/or VL domains, and encoding nucleicacid molecules and vectors according to the present invention may beprovided isolated and/or purified, e.g. from their natural environment,in substantially pure or homogeneous form, or, in the case of nucleicacid, free or substantially free of nucleic acid or genes of originother than the sequence encoding a polypeptide with the requiredfunction. Nucleic acid according to the present invention may compriseDNA or RNA and may be wholly or partially synthetic. Reference to anucleotide sequence as set out herein encompasses a DNA molecule withthe specified sequence, and encompasses a RNA molecule with thespecified sequence in which U is substituted for T, unless contextrequires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, yeast and baculovirus systemsand transgenic plants and animals. The expression of antibodies andantibody fragments in prokaryotic cells is well established in the art.For a review, see for example Pluckthun (1991). A common, preferredbacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a specific bindingmember for example Chadd & Chamow (2001), Andersen & Krummen (2002),Larrick & Thomas (2001). Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanomacells, YB2/0 rat myeloma cells, human embryonic kidney cells, humanembryonic retina cells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. ‘phage, as appropriate. For further details see,for example, Sambrook & Russell (2001). Many known techniques andprotocols for manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in Ausubel et al., 1988 and Ausubel et al., 1999.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture. Such a host cell may be in vivo. In vivopresence of the host cell may allow intracellular expression of thespecific binding members of the present invention as “intrabodies” orintracellular antibodies. Intrabodies may be used for gene therapy.

A still further aspect provides a method comprising introducing suchnucleic acid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay incorporated into the host cell or into an artificial chromosome.Incorporation may be either by random or targeted integration of one ormore copies at single or multiple loci. For bacterial cells, suitabletechniques may include calcium chloride transformation, electroporationand transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences that promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method that comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the followingexperimentation and the accompanying drawings, in which:

FIG. 1 shows concentration-inhibition curves for antibody neutralisationof human (FIG. 1A) and rat (FIG. 1B) NGF in the human TrkA receptorcalcium mobilisation assay. Human IgG4 NGF antibodies were compared withthe commercial NGF antibodies G1131, Mab5260Z, and MAB256 for inhibitionof intracellular calcium mobilisation evoked by 1 nM NGF. Data pointsindicate results from a single experiment and are mean ±sd of triplicatedeterminations for each antibody concentration.

FIG. 2 shows inhibition of intracellular calcium mobilisation in HEK-293cells recombinantly expressing the human TrkA receptor.Potency-optimised human IgG4 antibodies were evaluated for inhibition ofresponses evoked by 1 nM human (FIG. 2A), rat (FIG. 2B), and mouse (FIG.2C) NGF. Data points indicate results from a single experiment and aremean ±sd of triplicate determinations for each antibody concentration.

FIG. 3 shows the inhibitory effect of human IgG4 NGF antibodies in thePC12 cell survival assay. Cell survival was maintained by the presenceof 1 nM human NGF (FIG. 3A) or rat NGF (FIG. 3B). Data indicate mean ±sdfor triplicate determinations from a single experiment.

FIG. 4 shows inhibition of NGF-mediated TF-1 cell proliferation bygermline and non-germline human IgG4 NGF antibodies, and the referenceNGF antibody, MAB256. Cells were stimulated with 200 pM human NGF (FIG.4A), rat NGF (FIG. 4B), or mouse NGF (FIG. 4C). Data represent the mean±sem of triplicate determinations from a single experiment.

FIG. 5 shows the lack of cross-reactivity of the human IgG4 NGF antibody1252A5 with the neurotrophins BDNF, NT-3 and NT-4. 100 ng neurotrophinwas adsorbed per well. Each data point represents the mean ±sd oftriplicate determinations from a single experiment.

FIG. 6 shows saturation binding curves for human ¹²⁵I-NGF binding to thehuman IgG4 NGF antibodies 1252A5 (FIG. 6A) and G1152H5 (FIG. 6B).Calculated Kd values are 0.35 nM and 0.37 nM, respectively, and are theresult of a single experiment.

FIG. 7 shows saturation binding curves for rat ¹²⁵I-NGF binding to thehuman IgG4 NGF antibodies 1252A5 (FIG. 7A) and G1152H5 (FIG. 7B).Calculated Kd values are 0.44 nM and 0.50 nM, respectively, and are theresult of a single experiment.

FIG. 8 shows concentration-dependent inhibition of human (FIG. 8A) orrat (FIG. 8B) ¹²⁵I-NGF binding to TrkA receptor fusion protein, by humanIgG4 or reference antibodies. The concentration of radiolabelled NGF ineach assay well was approximately 150 pM. Data indicate the result of asingle experiment. See also Tables 6 and 7.

FIG. 9 shows concentration-dependant inhibition of human (FIG. 9A) orrat (FIG. 9B) ¹²⁵I-NGF binding to p75 receptor fusion protein, by humanIgG4 or reference antibodies. The concentration of radiolabelled NGF ineach assay well was approximately 150 pM. Data indicate the result of asingle experiment. See also Tables 8 and 9.

FIG. 10 shows dose-related inhibition of carrageenan-induced thermalhyperalgesia in the mouse, 48 h after systemic administration of thehuman IgG4 anti-NGF, 1252A5.

EXAMPLE 1 Isolation of Anti-NGF scFv

ScFv Antibody Repertoire

Three large single chain Fv (scFv) human antibody libraries cloned intoa phagemid vector, were used for selections. The libraries were derivedfrom (A) spleen lymphocytes (Hutchings, 2001), (B) a combination ofperipheral blood lymphocytes, tonsil B cells and bone marrow B cells(Vaughan et al., 1996) and (C) the light chains and VH CDR3 regions of Acombined with the framework of the DP47 germline heavy chain.

Selection of scFv

The phage selection procedure used was essentially as described inHutchings, supra. ScFv that recognised NGF were isolated from phagedisplay libraries in a series of selection cycles on human and rat NGF.In brief, unmodified antigen was coated to Nunc Maxisorb tubes. Phagewere incubated on the antigen in a total volume of 500 μl for 1 h priorto washing to remove unbound phage. Bound phage were then rescued asdescribed by Vaughan et al., supra and the selection process repeated.To assist the isolation of human/rodent cross-reactive scFv alternaterounds of selection were performed on the respective species isoforms ofNGF. A maximum of four rounds of selection was performed with any onelibrary using this alternating isoform selection strategy. Either humanor rat β-NGF were used as the initial antigen for first roundselections.

Outputs from rounds 2-4 were prioritised for biochemical screening basedon the percentage of NGF-specific clones isolated and the sequencediversity of these clones. The percentage of NGF-specific clones wasdetermined in each case by phage ELISA. Sequence diversity wasdetermined by DNA sequencing.

Phage ELISA Protocol

Cultures of phage transduced bacteria were prepared in 1 ml 2×TY mediumcontaining 100 μg/ml ampicillin and 50 μg/ml kanamycin with shaking at30° C. for 16 h. Phage supernatant was prepared by centrifugation of theculture (10 min at 3000 rpm) and blocking with 3% w/v milk powder in PBSfor 1 h. Blocked phage were then added to plates previously coated with(1 μg/ml) antigen or irrelevant antigen. Plates were washed between eachstep with three rinses in PBS-Tween 20 (0.1% v/v) followed by threerinses in PBS. Bound phage were detected by incubation with horseradishperoxidase (HRP)/anti-M13 conjugate (Amersham UK) diluted 1:5000 in 3%w/v milk powder PBS for 1 h, and developed by incubation withtetramethylbenzidine (TMB) substrate (Sigma). The colorimetric reactionwas stopped after an appropriate period by adding 0.5M sulphuric acid.Absorbance readings were taken at 450 nm. Clones which boundspecifically to the antigen were identified as having a signal on theantigen greater than or equal to 5× that on the irrelevant antigen.

DNA Sequencing

Double stranded DNA template for sequencing was obtained by PCR of scFvusing the primers FDTETSEQ24 (TTTGTCGTCTTTCCAGACGTTAGT—SEQ ID NO: 534)and PUCreverse (AGCGGATAACAATTTCACACAGG—SEQ ID NO: 535). Excess primerand dNTPs from the primary PCR were removed using Macherey-NagelNucleofast 96 well PCR plates (Millipore) according to themanufacturer's recommendations. VH genes were sequenced with primer Lseq(GATTACGCCAAGCTTTGGAGC SEQ ID NO: 536). VL genes were sequenced withprimer MYC Seq 10 (CTCTTCTGAGATGAGTTTTTG SEQ ID NO: 537). Each reactionmixture contained 20-40 ng DNA, 3-20 pmol primer and 4 μl Big DyeTerminator V3.0 (Applied Biosystems, UK) in a volume of 20 μl.Sequencing reactions consisted of 25 cycles of 96° C. 10 s; 50° C., 5 s;60° C., 4 min. Samples were run and analysed on an Applied Biosystems3700 DNA Analyser. Areas of ambiguity were analysed manually usingContinuity software developed in-house (Cambridge Antibody Technology,UK) and SeqEd DNA sequence manipulation software (Applied Biosystems,UK).

Biochemical Screen for NGF-Neutralising scFv

The output from the phage selection process was further screened toidentify clones that inhibited NGF binding to a TrkA receptorextracellular domain fusion protein.

Crude scFv samples were prepared from periplasmic lysates of E. coliTG-1 bacteria transfected with selected phage for evaluation in thebinding assay. Nunc Maxisorb 96 well plates were coated overnight withhuman TrkA receptor extracellular domain fusion protein (R&D Systems;coating concentration; human NGF assay, 0.25 nM; rat NGF assay, 1 nM).Assay plates were washed with PBS/Tween 20, blocked for 2 h using 1%bovine serum albumin (BSA) in PBS, and washed again. ScFv samples werepreincubated for 30 min with 1 nM human or rat recombinant β-NGF (R&DSystems) in 1% BSA. Samples were transferred in a volume of 100 μl toassay plates and incubated for 60 min at room temperature. Plates werewashed and NGF that remained bound to the plates was labelled using 0.3μg/ml of either anti-human NGF biotin (Peprotech) or anti-rat NGF biotin(R&D Systems) diluted in 1% BSA, followed by incubation for 60 min atroom temperature. Biotin-labelled anti-NGF was detected using the DELFIA(Wallac) time-resolved fluorescence detection system. Briefly, plateswere washed and 100 μl streptavidin Eu³⁺ added to each well, diluted1/1000 in DELFIA assay buffer. Plates were incubated for a further 60min at room temperature and washed with DELFIA wash buffer, followed byaddition of 100 μl DELFIA enhancement solution to each well. Plates wereread using a Wallac Victor fluorimetric plate reader (excitationwavelength 314 nm; emission wavelength 615 nm).

Clones that inhibited both human and rat NGF binding by more than 70% asperiplasmic lysate scFv preparations were re-evaluated as purified scFvin the binding assay. Purified scFv preparations were prepared asdescribed in Example 3 of WO01/66754. Protein concentrations of purifiedscFv preparations were determined using the BCA method (Pierce).Re-assay highlighted the following scFv antibodies that were potentneutralisers of human and rat NGF binding to the human TrkA receptorextracellular domain fusion protein:

1064F8 (VH SEQ ID NO: 2; VL SEQ ID NO: 7),

1022E3 (VH SEQ ID NO: 12; VL SEQ ID NO: 17),

1083H4 (VH SEQ ID NO: 22; VL SEQ ID NO: 27),

1021E5 (VH SEQ ID NO: 32; VL SEQ ID NO: 37),

1033G9 (VH SEQ ID NO: 42; VL SEQ ID NO: 47),

1016A8 (VH SEQ ID NO: 52; VL SEQ ID NO: 57),

1028F8 (VH SEQ ID NO: 62; VL SEQ ID NO: 67),

1033B2 (VH SEQ ID NO: 72; VL SEQ ID NO: 77),

1024C4 (VH SEQ ID NO: 82; VL SEQ ID NO: 87), and

1057F11 (VH SEQ ID NO: 92; VL SEQ ID NO: 97).

EXAMPLE 2 Expression of Human IgG4 Antibodies and In Vitro FunctionalEvaluation of NGF-Neutralising Potency

The NGF-neutralising scFv 1064F8, 1022E3, 1083H4, 1021E5, 1033G9,1016A8, 1028F8, 1033B2, 1024C4, and 1057F11 were reformatted as humanIgG4 antibodies and assayed for NGF neutralising potency in a whole-cellassay system.

IgG Conversion

Vectors were constructed for the most potent scFv clones to allowre-expression as whole antibody human IgG4, essentially as described byPersic et al. (1997). EBNA-293 cells maintained in conditioned mediumwere co-transfected with constructs expressing heavy and light chaindomains. Whole antibody was purified from the medium using protein Aaffinity chromatography (Amersham Pharmacia). The purified antibodypreparations were sterile filtered and stored at 4° C. in phosphatebuffered saline (PBS) prior to in vitro potency evaluation. Proteinconcentration was determined spetrophotometrically according to Mach etal. (1992).

FLIPR Assay of Intracellular Calcium Mobilisation

The potency and efficacy of anti-human IgGs for neutralising NGF weredetermined in a cell-based fluorescent calcium-mobilisation assay. Thepotency of human antibodies was compared with mouse anti-human NGF(MAB256; R&D Systems), rat anti-mouse NGF (G1131; Promega), and mouseanti-mouse NGF (MAB5260Z; Chemicon). Anti-NGF IgGs were co-incubatedwith recombinant human β-NGF (Calbiochem, 480275) or recombinant ratβ-NGF (R&D Systems, 556-NG-100). The complex was then added to HEK293cells expressing recombinant human TrkA receptors loaded with thecalcium sensitive dye, Fluo-4, and then Ca²⁺-dependent fluorescence wasmonitored.

HEK cells (peak-S, Edge Biosystems) transfected with recombinant humanTrkA (obtained from M. Chao, Skirball Institute, NY) were grown inDulbecco's Modified Eagle's Medium (MEM, Cellgro, MT10-017-CV)supplemented with 10% fetal bovine serum (Hyclone, SH30071.03), 1.5μg/ml puromycin (Edge Biosystems, 80018) and 1% penicillin-streptomycin.Confluent cells were harvested by dislodging the cells with Dulbecco'sphosphate buffered saline (DPBS), and then loaded with loading buffercontaining 6 μM Fluo-4 (Molecular Probes) at 37° C. for 1.5 h in thepresence of an anion transport inhibitor (2.5 mM probenecid in 1%FBS/MEM). After washing the cells once with assay buffer (2.5 mMprobenecid in 0.1% BSA Hank's/HEPES), the cells were plated onpoly-D-lysine coated, clear bottom 96-well plates (Costar #3904) atapproximately 60,000 cells/well in 120 μl. The cells were incubated inthe dark for 30 min at room temperature, and the plates were thencentrifuged at 1,200 rpm (290×g) for 5 min. Prior to testing, the plateswere pre-warmed at 35° C. for 20 min. Test IgGs were assayed at 7concentrations in triplicate wells. Thirty-five microlitres of 10×anti-NGF IgG and equal amounts of 10 nM human-, rat- or mouse 2.5 S NGFwere pre-incubated at room temperature for approximately 1 h. Platescontaining the pre-complexed IgGs and plates containing 100 μl/wellassay buffer were pre-warmed at 35° C. for 20 min before testing on theFluorometric Imaging Plate Reader (FLIPR; Molecular Devices). Followingthe addition of 80 μl/well assay buffer to the cell plate and incubationfor 5 min at 35° C., 50 μl/well of diluted anti-human IgG/NGF complexwas added to the cell plate in the FLIPR with continuous monitoring ofthe Ca²⁺-dependent fluorescence. The NGF-induced fluorescence wascalculated as the difference between the baseline fluorescence intensityjust prior to NGF addition and highest fluorescence intensity attainedin 2 minutes following NGF addition. The average of triplicateNGF-induced fluorescence values in the absence of antibody was definedas 100% calcium mobilisation. In the presence of antibody, the average±SD of triplicate NGF-induced fluorescence values was calculated as apercent of control calcium mobilisation. The percent of control calciummobilisation values were plotted as function of the log of the IgGconcentration. IC₅₀ values were calculated by fitting the sigmoidaldose-response (variable slope) function using Prism (GraphPad).

All antibodies tested displayed concentration-related inhibition ofhuman NGF-evoked intracellular calcium mobilisation. The rank order ofpotency for neutralisation of human NGF was1064F8>1022E3>1083H4≧1033G9≧1016A8≧1028F8≧1021E5≧1033B2>>1024C4>>1057F11(Table 1). Five antibodies were further evaluated for functionalneutralisation of rat NGF, and these were approximately equipotentagainst both species isoforms (FIGS. 1A and 1B; Table 1).

EXAMPLE 3 Isolation of Optimised Human IgG4 NGF Antibodies

Ribosome Display scFv Potency Optimisation

Large ribosome display libraries were created and selected for scFv thatspecifically recognised recombinant human NGF (R&D Systems), essentiallyas described in Hanes et al. (2000). Initially, the clones 1064F8,1022E3, 1083H4, 1021E5, 1033G9, 1016A8, 1028F8, 1033B2, and 1024C4 wereconverted to ribosome display format, where the templates weresubsequently used for library creation. The clone 1057F11 was not chosenfor potency optimisation, and therefore a ribosome display template wasnot made for this antibody. On the DNA level, a T7 promoter was added atthe 5′-end for efficient transcription to mRNA. On the mRNA level, theconstruct contained a prokaryotic ribosome-binding site (Shine-Dalgarnosequence). At the 3′ end of the single chain, the stop codon was removedand a portion of gIII was added to act as a spacer (Hanes et al.,supra).

Ribosome display libraries derived from 1064F8, 1022E3, 1083H4, 1021E5,1033G9, 1016A8, 1028F8, 1033B2, and 1024C4 were created by mutagenesisof the scFv HCDR3. PCR reactions were performed with non-proof readingTaq polymerase. Affinity-based selections were performed whereby,following incubation with the library, biotinylated human NGF wascoupled to streptavidin-coated paramagnetic beads (Dynal M280). Boundtertiary complexes (mRNA-ribosome-scFv) were recovered by magneticseparation whilst unbound complexes were washed away. The mRNA encodingthe bound scFv were then rescued by RT-PCR as described in Hanes et al.,(supra) and the selection process repeated with decreasingconcentrations (100 nM-10 pM over five rounds) of biotinylated human NGFpresent during the selection.

Error-prone PCR was also used to further increase library size. An errorrate of 7.2 mutations per 1,000 bp was employed (Diversify™, Clontech)during the selection regime. Error-prone PCR reactions were performedbefore selections commenced at rounds three and four using biotinylatedhuman NGF concentrations of 1 nM and 0.1 nM, respectively.

A representative proportion of scFv from the output of selection roundsthree, four and five were ligated into pCantab6 vector (Vaughan et al.,1996) and cloned in the TG1 strain of E. coli. A sample of these scFvwas DNA sequenced as described in Example 1 to confirm sequencediversity of the output before screening in vitro for NGF neutralisingactivity. Clones were screened as unpurified scFv in the NGF/TrkAreceptor extracellular domain fusion protein binding assay, as describedin Example 1. The concentration of the periplasmic lysate scFvpreparations in the assays was reduced to 0.5%-5% of the final assayvolume, and clones that inhibited both human and rat NGF binding >95%were isolated for further study. In this way a panel ofpotency-optimised, cross-reactive NGF neutralisers was isolated.Surprisingly, the most potent NGF neutralisers were derived from theparent clones 1021E5 and 1083H4, which were not the most potent of theparent antibodies. Optimised clones were sequenced and reassayed aspurified scFv to confirm potency before reformatting to human IgG4 asdescribed in Example 2.

Antibodies derived from the parent clone 1021E5 (VH SEQ ID NO: 32; VLSEQ ID NO: 37) and converted to human IgG4 format were 1126F1 (VH SEQ IDNO: 102; VL SEQ ID NO: 107), 1126G5 (VH SEQ ID NO: 112; VL SEQ ID NO:117), 1126H5 (VH SEQ ID NO: 122; VL SEQ ID NO: 127), 1127D9 (VH SEQ IDNO: 132; VL SEQ ID NO: 137), 1127F9 (VH SEQ ID NO: 142; VL SEQ ID NO:147), 1131D7 (VH SEQ ID NO: 152; VL SEQ ID NO: 157), 1131H2 (VH SEQ IDNO: 162; VL SEQ ID NO: 167), 1132A9 (VH SEQ ID NO: 172; VL SEQ ID NO:177), 1132H9 (VH SEQ ID NO: 182; VL SEQ ID NO: 187), 1133C11 (VH SEQ IDNO: 192; VL SEQ ID NO: 197), 1134D9 (VH SEQ ID NO: 202; VL SEQ ID NO:207), 1145D1 (VH SEQ ID NO: 212; VL SEQ ID NO: 217), 1146D7 (VH SEQ IDNO: 222; VL SEQ ID NO: 227), 1147D2 (VH SEQ ID NO: 232; VL SEQ ID NO:237), 1147G9 (VH SEQ ID NO: 242; VL SEQ ID NO: 247), 1150F1 (VH SEQ IDNO: 252; VL SEQ ID NO: 257), 1152H5 (VH SEQ ID NO: 262; VL SEQ ID NO:267), 1155H1 (VH SEQ ID NO: 272; VL SEQ ID NO: 277), 1158A1 (VH SEQ IDNO: 282; VL SEQ ID NO: 287), 1160E3 (VH SEQ ID NO: 292; VL SEQ ID NO:297), 1165D4 (VH SEQ ID NO: 302; VL SEQ ID NO: 307), 1175H8 (VH SEQ IDNO: 312; VL SEQ ID NO: 317), 1211G10 (VH SEQ ID NO: 322; VL SEQ ID NO:327), 1214A1 (VH SEQ ID NO: 332; VL SEQ ID NO: 337), 1214D10 (VH SEQ IDNO: 342; VL SEQ ID NO: 347), 1218H5 (VH SEQ ID NO: 352; VL SEQ ID NO:357), and 1230H7 (VH SEQ ID NO: 362; VL SEQ ID NO: 367).

Antibodies derived from the parent clone 1083H4 (VH SEQ ID NO: 22; VLSEQ ID NO: 27) and converted to human IgG4 format were 1227H8 (VH SEQ IDNO: 372; VL SEQ ID NO: 377) and 1230D8 (VH SEQ ID NO: 382; VL SEQ ID NO:387).

Germlining Framework Regions of 1133C11 to Derive 1252A5 and Other1021E5 Variants

Examination of the VH and VL CDR sequence information for optimisedclones derived from 1021E5 highlighted that a large proportion of theseantibodies contained the amino acid sequence LNPSLTA (SEQ ID NO: 531) inVH CDR3 (i.e. amino acids 100A to 100G according to the Kabat numberingsystem). These clones are shown in Table 2a, highlighting how they varyin amino acid sequence in the VH and VL CDR regions, together with anestimate of their NGF neutralising potency when assayed as purifiedscFv. Of these clones, 1133C11 differed in CDR regions from 1021E5 onlyby the 7 consecutive amino acids 100A to 100G as described. Therefore,1133C11 was chosen for germlining, first to confirm that potency wasretained with the modified framework, and second to allow subsequent CDRmutations to be introduced, if desired, in order to generate furthergermline antibodies of interest from the same lineage.

The derived amino acid VH and VL sequences of 1133C11 were aligned tothe known human germline sequences in the VBASE database (Tomlinson[1997], MRC Centre for Protein Engineering, Cambridge, UK) and theclosest germline identified. The closest germline for the VH of 1133C11was identified as DP10, a member of the VH1 family. The 1133C11 VH has 5amino acid changes from the DP10 germline within framework regions. Theclosest germline for the VL of 1133C11 was identified as DPL5, a memberof the Vλ1 family. The 1133C11 VL has only 4 changes from the germlinewithin framework regions. Framework regions of 1133C11 were returned togermline by site directed mutagenesis of the scFv to derive the scFv1252A5 (VH SEQ ID NO: 392; VL SEQ ID NO: 397). This was converted tohuman IgG4 as described in Example 2. Germlining of Other Variants of1021E5-derived clones was achieved by introducing CDR mutations onto thegermlined 1252A5 IgG4 backbone. This method resulted in generation ofthe germlined antibodies G1152H5 (VH SEQ ID NO: 402; VL SEQ ID NO: 407),G1165D4 (VH SEQ ID NO: 412; VL SEQ ID NO: 417) and G1230H7 (VH SEQ IDNO: 422; VL SEQ ID NO: 427).

EXAMPLE 4 Evaluation of Optimised Human IgG4 Antibodies in the FLIPRAssay of Intracellular Calcium Mobilisation

Optimised human IgG4 NGF antibodies were evaluated in an assay ofNGF-evoked intracellular calcium mobilisation in cells recombinantlyexpressing the human TrkA receptor, as described in Example 2.Antibodies were assayed for neutralising activity against human, rat,and mouse NGF (FIGS. 2A, 2B, and 2C; Table 3).

Intracellular calcium mobilisation evoked by 1 nM NGF was inhibited byall optimised human antibodies tested. Optimised antibodies displayedsubnanomolar IC₅₀ values, in most cases representing greater than onehundredfold enhancement of NGF neutralising potency over the parent IgGs(Table 3). Neutralising potencies (IC₅₀) of the germlined human IgG4antibodies against the human, rat and mouse NGF isoforms were,respectively:

-   -   1252A5—0.33 nM, 0.29 nM, and 0.26 nM;    -   G1152H5—0.22 nM, 0.27 nM, and 0.18 nM;    -   G1165D4—0.32 nM, 0.33 nM, and 0.27 nM;    -   G1230H7—0.31 nM, 0.34 nM, and 0.25 nM.

These results highlight the efficiency and value of the ribosome displaytechnique for antibody potency optimisation. A more conventionalapproach to antibody optimisation in the past has been to generate phagedisplay libraries of variant scFv antibodies. This process is labourintensive and slower than the ribosome display method, which often meansthat only a single parent scFv is used as the starting point for libraryconstruction. The relative ease of generating ribosome display librariesallows multiple scFv parents to be optimised simultaneously and, asdemonstrated in Example 3, this can lead to the isolation of highlypotent antibodies derived from parent clones that would have beenotherwise overlooked for optimisation.

EXAMPLE 5

Evaluation of Optimised Human IgG4 Antibodies in a PC12 Cell SurvivalAssay

In the PC12 assay, NGF maintains the survival of serum-deprived rat PC12cells expressing native TrkA and p75 receptors for two days.Neutralising NGF antibodies reduce cell survival measured withAlamarBlue.

Rat pheochromocytoma PC12 cells were grown in RPMI 1640 (Cellgro,18040181) supplemented with 5% fetal bovine serum (JRH, 12103-78P), 10%heat-inactivated donor horse serum (JRH, 12446-77P), and 1%penicillin-streptomycin. The cells were harvested by trituration, andthen washed twice with serum-free RPMI 1640 containing 0.01% BSA (Sigma,A7030). Cells were plated in rat tail collagen (Biological TechnologyInstitute, BT-274)-coated 96 well plates at 50,000 cells/well in 120 μlserum-free media with 0.01% BSA. Serial dilutions of 5× anti-human NGFIgGs were made using serum-free media and 40 μl/well was added to thecell plate. 40 μl/well 0.5 nM human β-NGF (Calbiochem, 480275) or ratrecombinant NGF (R&D Systems, 556-NG-100) was added to the plate, andthe total volume was brought up to 200 μl/well with serum-free medium.Maximal cell death was defined by 80 μl/well of serum-free medium intriplicate wells. 100% survival was defined by 40 μl/well serum-freemedia and 40 μl/well 0.5 nM NGF in triplicate wells. The plates wereincubated at 37° C. in 5% CO₂ for 48 h.

To measure the cell viability, 22 μl/well AlamarBlue was added and theplates were read immediately to determine the background fluorescence ineach well with a fluorometric plate reader (BMG) at 530 nm excitationwavelength and 590 nm emission wavelength. Following an incubation for6-7 h at 37° C., the plate was re-read to determine total fluorescence.The Alamar blue fluorescence was calculated as the difference betweenthe background and total fluorescence intensities. In the presence ofantibody, the average ±SD of triplicate fluorescence values wascalculated as a percent of the average of triplicate 100% survivalfluorescence values. The percent of control survival values were plottedas function of the log of the IgG concentration. IC₅₀ values werecalculated by fitting the sigmoidal dose-response (variable slope)function using Prism (GraphPad).

Results from the PC12 assay further confirmed the increased potency ofoptimised human IgG4 NGF antibodies over their parent IgGs. Antibodiesinhibited human and rat NGF-maintained PC12 cell survival in aconcentration-related manner (FIGS. 3A and 3B; Table 3). Germliningappeared to reduce the NGF neutralising potency of the test antibodies,particularly against the rat NGF isoform. For example, the mean IC₅₀ ofthe germline antibody G1152H5 for inhibition of cell survival mediatedby 1 nM human or rat NGF was 1.1 nM and 7.3 nM, respectively. Incontrast, the mean IC₅₀ for neutralisation of 1 nM human or rat NGF by1152H5 (ie non-germline antibody) was 0.40 nM and 0.38 nM, respectively.

EXAMPLE 6 NGF-Neutralising Activity in a TF-1 Cell Proliferation Assay

The TF-1 cell line is a human premyeloid cell line that can bestimulated to proliferate by exogenous growth factors and cytokines.TF-1 cells express the human TrkA receptor, and proliferate in responseto activation with NGF. The TF-1 cell proliferation assay was used tofurther characterise the in vitro functional potency of neutralisinghuman IgG4 NGF antibodies.

TF-1 cells were obtained from R&D Systems and maintained according tosupplied protocols. Assay media comprised of RPMI-1640 with GLUTAMAX I(Invitrogen) containing 5% foetal bovine serum (Hyclone) and 1% sodiumpyruvate (Sigma). Prior to each assay, TF-1 cells were pelleted bycentrifugation at 300×g for 5 minutes, the media removed by aspirationand the cells resuspended in assay media. This process was repeatedthree times with cells resuspended at a final concentration of 10⁵/ml inassay media and 100 μl was added to each well of a 96 well flat bottomedtissue culture assay plate to give final cell density at 1×10⁴/well.Test solutions of antibodies (in triplicate) were diluted to give afinal assay concentration of 1 μg/ml in assay media and titrated 1:5across assay plate. An irrelevant antibody (CAT-001) not directed atNGF, was used as a negative control. In addition, a reference monoclonalantibody MAB256 (R&D Systems) was used as a positive control. Fiftymicrolitres of test antibodies were then added to each well followed by5011 of native purified murine (7S form; Invitrogen), rat (Sigma) orhuman (Sigma) NGF diluted to give a final assay concentration of 200 μM.Assay plates were incubated for 68 h at 37° C. in 5% CO₂ in a humidifiedchamber. Twenty microlitres of tritiated thymidine (5.0 μCi/ml, NEN) wasthen added to each assay well and assay plates were returned to theincubator for a further 5 h. Cells were harvested onto 96 well glassfibre filter plates (Perkin Elmer) using a cell harvester. MicroScint20™ (50 μl) was then added to each well of the filter plate and[³H]-thymidine incorporation quantified using a Packard TopCountmicroplate liquid scintillation counter. In the presence of antibody,thymidine incorporation (quantified as counts per minute) was calculatedas the difference between the average background (i.e. cells not exposedto NGF) and the average total (i.e. cells stimulated with NGF) countsper minute and expressed as a percent of maximum proliferation. Thepercent maximum proliferation was plotted as function of the log of theIgG concentration. IC₅₀ values were calculated by fitting the sigmoidaldose (variable slope) function using GraphPad prism.

Human IgG4 NGF antibodies were potent inhibitors of TF-1 cellproliferation mediated by human, rat and mouse NGF isoforms (FIGS. 4A,4B, and 4C). These results demonstrate that antibodies derived from the1021E5 lineage can disrupt NGF signalling mediated by activation ofnative human NGF receptors in vitro. In accordance with the observedactivity of human NGF antibodies in the PC12 cell survival assay(Example 5), non-germline antibodies were more potent than theirgermline counterparts in the TF-1 proliferation assay (Table 3). Basedon mean IC50 data, the rank order of potency of the antibodies testedfor inhibition of proliferation mediated by 200 pM human NGF was1133C11>1152H5>1252A5=G1152H5>>MAB256.

EXAMPLE 7 Cross-Reactivity of Anti-NGF IgGs with Other Neurotrophins

ELISAs were performed to determine the cross-reactivity of the anti-NGFIgGs for other neurotrophins. The ELISAs consisted of coating plateswith 100 ng/well human NGF (R&D systems, 256-GF), brain derivedneurotrophic factor (BDNF; R&D systems, 248-BD), neurotrophin-3 (NT-3;R&D systems, 257-N3), or neurotrophin-4 (NT4; R&D systems, 257-N4) atroom temperature for 5-6 h, followed by blocking the plates with 0.25%HSA at 4° C. overnight. Increasing concentrations of anti-NGF IgG,ranging from 0.03-10 nM, were incubated at room temperature for 2 h toallow binding to each neurotrophin. Anti-NGF IgGs were detected with abiotinylated anti-human polyclonal antibody (1:300) (Rockland 609-1602),streptavidin-linked alkaline phosphatase (1:1000), and fluorescentSubstrate A. Positive controls demonstrating neurotrophin binding to theplate utilised commercial biotinylated anti-human polyclonal antibodies(R&D Systems anti-NGF BAF 256, anti-BDNF BAM 648, anti-NT-3 BAF 267,anti-NT-4 BAF 268), which were detected directly usingstreptavidin-linked alkaline phosphatase and subsequent addition ofSubstrate A. Nonspecific binding was determined using wells coated withBSA instead of neurotrophin. Product development was followed over timefrom 0-60 min after addition of Substrate A. Anti-NGF IgG 1064F8 wasused to optimise the assay. For 1064F8, there was linear productdevelopment for 15 min, which then leveled off with time, probably dueto substrate depletion. Cross-reactivities were calculated as a percentof the specific binding to neurotrophin relative to NGF for allconcentrations of IgG. For high affinity IgGs such as 1064F8, percentcross-reactivities were calculated using 15 min product developmentdata. For low affinity IgGs such as 1016A8, percent cross-reactivitieswere calculated using 60 min product development data.

The cross-reactivities of seven human IgG4 NGF antibodies to BDNF, NT-3,and NT-4 relative to NGF were determined. At the concentrations tested,all seven antibodies showed negligible cross-reactivity (Table 4). Forexample, with 1252A5 the highest levels of cross-reactivity observedwith NT-3, NT-4 and BDNF were 1.1%, 0.9% and 1.4%, respectively (FIG.5).

EXAMPLE 8 Determination of the NGF-Binding Affinity of Human NGFAntibodies

The NGF-binding affinities of human IgG4 NGF antibodies were determinedusing a radioligand-binding assay format, performed at room temperature.Briefly, flashplates (Perkin Elmer SMP200) were coated with 100 μl/wellof 2.2 μg/ml goat anti-human IgG (Sigma-Aldrich, UK) in phosphatebuffered saline (PBS) for 1 h. Wells were washed with PBS and thenblocked for 1 h with 200 μl/well PBS containing 3% w/v bovine serumalbumin (BSA; Sigma-Aldrich, UK). Wells were washed with PBS and 10 ngof human NGF antibody was added to each well in a volume of 0.1 ml PBScontaining 0.5% w/v BSA. Following incubation for 1 h plates were washedwith PBS.

Radioiodinated human and rat NGF were obtained from Amersham UK (human¹²⁵I-NGF, Amersham cat. no. IM286; rat ¹²⁵I-NGF, custom-labelledrecombinant rat β-NGF purchased from R&D Systems, cat. no. 556-GF-100).Each ¹²⁵I-NGF isoform was serially diluted in assay buffer (PBScontaining 0.5% w/v BSA and 0.05% v/v Tween 20) and duplicate 100 μlsamples were added to the assay plate to give a measure of ‘totalbinding’ over the concentration range 2 μM-15 nM. Non-specific binding(NSB) was determined at each ¹²⁵I-NGF concentration by measuring bindingin the presence of a large excess of non-radiolabelled NGF. NSB wellscontained ¹²⁵I-NGF (2 pM-15 nM) together with a final concentration of500 nM unlabelled human β-NGF (R&D Systems Cat. No. 256-GF-100) or ratβ-NGF (R&D Systems Cat. No. 556-GF-100), as appropriate. Plates wereincubated overnight, and wells counted for 1 min on a gamma counter(TopCount NXT, Perkin Elmer). Specific binding was calculated accordingto the formula ‘specific binding=total binding−non-specific binding’.Binding curves were plotted and binding parameters determined accordingto a one-site saturation binding model using Prism software (GraphPadSoftware Inc., USA).

Human and rat ¹²⁵I-NGF displayed saturable, high affinity binding to thehuman IgG4 NGF antibodies, 1252A5 and G1152H5 (FIGS. 6 and 7).Calculated Kd values for the binding interaction with human ¹²⁵I-NGFwere 0.35 nM for 1252A5, and 0.37 nM for G1152H5. Kd values for rat¹²⁵I-NGF binding were 0.44 nM for 1252A5 and 0.50 nM for G1152H5.

EXAMPLE 9 Determination of Ki Values for Inhibition of NGF Binding toHuman TrkA and p75 Receptors

Experiments were performed to determine whether the antibodies 1252A5and G1152H5 display differential inhibition of NGF binding to TrkA andp75 receptors. Competition binding experiments were designed in order tocalculate binding inhibition constant (Ki) values. IC₅₀s were calculatedfor antibody-mediated inhibition of radiolabelled human or rat NGFbinding to human TrkA- or p75-receptor fusion proteins. Ki values werethen derived using the Cheng-Prusoff equation.

Human TrkA and p75 receptor fusion proteins (R&D Systems) were dilutedin Dulbecco's PBS (Gibco) to final concentrations of 10 nM and 0.6 nM,respectively. Maxisorp Nunc white 96 well microtitre plates (Nalge Nunc)were coated overnight at 4° C. with 100 μl/well of the diluted TrkA orp75 receptor solution. Plates were washed 3 times with PBS Tween 20, andthen blocked with 200 μl/well 3% w/v bovine serum albumin (BSA) in PBS.Plates were washed after 1 h incubation at room temperature. Testantibodies were diluted to the desired concentration in assay buffer(0.5% w/v BSA and 0.05% v/v Tween 20 in PBS). An irrelevant antibody,not directed towards NGF, was used as a negative control whilstnon-radiolabelled NGF was used as a reference inhibitor of radioligandbinding. Duplicate wells were prepared for each concentration of testsample. Human or rat ¹²⁵I-NGF (Amersham Biosciences) was diluted withassay buffer such that the final concentration in assay wells, whenmixed with test sample, was 150 μM in a total assay volume of 100 μl.Assay plates were incubated at room temperature for 2 h before washingwith PBS/Tween 20 to remove unbound ¹²⁵I-NGF. Bound radiolabel wasquantified by addition of 100 μl/well Microscint 20 (Perkin Elmer)followed by counting using a Packard TopCount microplate liquidscintillation counter. Data were plotted and analysed using GraphpadPrism software to calculate IC₅₀ values for each experiment, and toderive the corresponding Ki according to the Cheng-Prusoff equation;Ki=IC ₅₀/(1+D/Kd)Where:

-   D=the NGF concentration in the assay (nominally 150 μM, but actual    assay concentrations were determined for each experiment)-   Kd=the affinity of NGF for the TrkA or p75 receptor under identical    assay conditions. This was determined by saturation binding analysis    of ¹²⁵I-NGF binding to TrkA and p75 receptors in separate    experiments.

All antibodies evaluated, except the human IgG4 control, inhibited humanand rat ¹²⁵I-NGF binding to human TrkA and p75 receptors (FIGS. 8 and 9;Table 5). Table 6 shows IC50 and Ki values calculated from data shown inFIG. 8A; Table 7 shows IC50 and Ki values calculated from data shown inFIG. 8B; Table 8 shows IC50 and Ki values calculated from data shown inFIG. 9A; Table 9 shows IC50 and Ki values calculated from data shown inFIG. 9B. Antibody 1252A5 consistently showed the greatest potency of¹²⁵I-NGF binding inhibition. Interestingly, binding inhibition constantvalues determined for 1252A5-mediated inhibition of NGF binding to TrkAand p75 receptors were significantly different. Thus, the calculatedmean pKi for inhibition of human NGF binding to TrkA and p75 was10.26±0.08 and 9.85±0.04, respectively (P<0.01, Student's T-test; bothn=3). Calculated mean pKi for inhibition of rat NGF to TrkA and p75receptors was 9.79±0.04 and 9.55±0.03, respectively (P<0.05, Student'sT-test; both n=3). This result suggests that 1252A5 is a preferentialinhibitor of the interaction between NGF and the TrkA receptor, andunexpectedly contrasts with the results obtained with G1152H5 for whichthere was no significant difference between corresponding pKi values(Table 5).

EXAMPLE 10 Antihyperalgesic Activity of Human IgG4 NGF Antibodies

The antihyperalgesic activity of NGF antibodies was evaluated in a mousemodel of carrageenan-induced thermal hypersensitivity. Male mice (20-25g body weight) were initially acclimatised to the test apparatus for 2h. The following day, baseline measures of responsiveness to thermalstimulation of both hind paws were determined. A focussed heat sourcewas applied to the plantar hind paw surface, and the latency towithdrawal was recorded, according to the method of Hargreaves et al.(1988). Baseline values were calculated as the mean of triplicatedeterminations for each paw, recorded 10 min apart. Mice then receivedan intraperitoneal injection of NGF-neutralising human IgG4 antibody orcontrol isotype-matched null antibody in phosphate-buffered saline (PBS)vehicle. Twenty-four hours later, inflammatory hyperalgesia was inducedby subplantar injection of carrageenan (2% w/v in PBS; 30 μl injectionvolume). After a further 24 h period, withdrawal latencies were againdetermined for inflamed and non-inflamed hind paws.

Thermal hyperalgesia observed 24 h after carrageenan injection wasdose-dependently inhibited by pretreatment of mice with the human IgG4NGF antibody 1252A5 (FIG. 10).

All documents cited are incorporated herein by reference.

REFERENCES

-   Al-Lazikani, et al. Journal Molecular Biology (1997) 273(4), 927-948-   Aloe, L. and Tuveri, M. A. (1997) Clin Exp Rheumatol, 15(4): 433-8.-   Amann, R. and Schuligoi R. (2000) Neurosci Lett, 278(3): 173-6.-   Andersen D C and Krummen L (2002) Current Opinion in Biotechnology    13: 117-   Ausubel et al. eds., Short Protocols in Molecular Biology: A    Compendium of Methods from Current Protocols in Molecular Biology,    John Wiley & Sons, 4^(th) edition 1999-   Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates and    Radiopharmaceuticals 4: 915-922-   Barbas et al., 1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813-   Bennett, D. L. et al. (1998) Eur J Neurosci, 10(4): 1282-91.-   Bennett, D. L. (2001) Neuroscientist, 7(1): 13-7.-   Bergmann I. et al., Neurosci Lett., 255(2) 87-90, 1998-   Bird et al, Science, 242, 423-426, 1988;-   de Castro, F. et al. (1998) Eur J Neurosci, 10(1): 146-52.-   Chadd H E and Chamow S M (2001) Current Opinion in Biotechnology 12:    188-194-   Cho, H. J. et al. (1996) Brain Res, 716(1-2): 197-201.-   Chothia, et al. Science, 223, 755-758 (1986)-   Chothia C. et al. Journal Molecular Biology (1992) 227, 799-817    Current Protocols in Molecular Biology, Second Edition, Ausubel et    al. eds., John Wiley & Sons, 1988-   Denison David G. T. (Editor), Christopher C. Holmes, Bani K.    Mallick, Adrian F. M. Smith. Bayesian Methods for Nonlinear    Classification and Regression (Wiley Series in Probability and    Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369-   Fjell, J. et al. (1999) J Neurosci Res, 57(1): 39-47.-   Garaci, E. et al. (2003) Proc Natl Acad Sci USA, 100(15): 8927-8932.-   Ghose, Arup K. & Viswanadhan, Vellarkad N. Combinatorial Library    Design and Evaluation Principles, Software, Tools, and Applications    in Drug Discovery. ISBN: 0-8247-0487-8-   Gram et al., 1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580-   Guex, N. and Peitsch, M. C. Electrophoresis (1997) 18, 2714-2723-   Haan & Maggos (2004) BioCentury, 12(5): A1-A6-   Hanes et al., Methods in Enzymology, 328: 24, (2000)-   Hargreaves et al., Pain, 32: 77 (1988)-   Heumann, R. et al. (1987) J Cell Biol, 104(6): 1623-31.-   Holliger, P. and Winter G. Current Opinion Biotechnol 4, 446-449    1993-   Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993-   Holt et al (2003) Trends in Biotechnology 21, 484-490-   Hongo, J. S. et al. (2000) Hybridoma 19(3): 215-227.-   Hoyle, G. W. (2003) Cytokine Growth Factor Rev, 14(6): 551-8.-   Hu, S. et al, Cancer Res., 56, 3055-3061, 1996.-   Huang, E. J. and Reichardt, L. F. (2001) Ann Rev Neurosci, 24:    677-736.-   Huston et al, PNAS USA, 85, 5879-5883, 1988-   Hutchings, in Antibody Engineering, Konterman and Dubel eds.,    Springer, Berlin, pp. 93-108 [2001]-   Indo, Y. (2002) Clin Auton Res, 12 Suppl 1: I20-32.-   Jagger, S. I. et al. (1999) Br J Anaesth, 83(3): 442-8.-   Kabat, E. A. et al, Sequences of Proteins of Immunological Interest.    4^(th) Edition. US Department of Health and Human Services. 1987-   Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological    Interest, 5th Edition. US Department of Health and Human Services,    Public Service, NIH, Washington.-   Kandel, Abraham & Backer, Eric. Computer-Assisted Reasoning in    Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN:    0133418847-   Kay, B. K., Winter, J., and McCafferty, J. (1996) Phage Display of    Peptides and Proteins: A Laboratory Manual, San Diego: Academic    Press. Knappik et al. J. Mol. Biol. (2000) 296, 57-86-   Koide et al. (1998) Journal of Molecular Biology, 284: 1141-1151.-   Koltzenburg, M. et al. (1999) Eur J Neurosci, 11(5): 1698-704.-   Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New    York, LLC; 2001, ISBN: 3540413545.-   Krebs et al. Journal of Immunological Methods 254 2001 67-84-   Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's    Perspective (Oxford Statistical Science Series, No 22 (Paper)).    Oxford University Press; (December 2000), ISBN: 0198507089-   Larrick J W and Thomas D W (2001) Current Opinion in Biotechnology    12:411-418.-   Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664-   Lommatzsch, M. et al. (2003) Ann NY Acad Sci, 992: 241-9.-   Lowe, E. M. et al. (1997) Br J Urol, 79(4): 572-577.-   Ma, Q. P. and Woolf, C. J. (1997) Neuroreport, 8(4): 807-10.-   Mach et al., Analytical Biochemistry, 200: 74, (1992)-   Mamet, J. et al. (2003) J Biol Chem, 278(49): 48907-13.-   Marks et al Bio/Technology, 1992, 10:779-783-   McArthur, J. C. et al. (2000) Neurology, 54(5): 1080-8.-   McCafferty et al (1990) Nature, 348, 552-554-   Mendell, L. M. and Arvanian, V. L. (2002) Brain Res Rev, 40(1-3):    230-9.-   Mendez, M. et al. (1997) Nature Genet, 15(2): 146-156.-   Nakagawara, A. (2001) Cancer Lett, 169(2): 107-14.-   Norman et al. Applied Regression Analysis. Wiley-Interscience;    3^(rd) edition (April 1998) ISBN: 0471170828-   Nygren et al. (1997) Current Opinion in Structural Biology, 7:    463-469.-   Owolabi, J. B. et al. (1999) J Pharmacol Exp Ther, 289(3): 1271-6.-   Persic et al., Gene, 187: 9, (1997)-   Petty, B. G. et al. (1994) Ann Neurol, 36(2): 244-6.-   Pleuvry B. & Pleuvry A., (2000) Analgesia: Markets and Therapies,    ISBN 1860674143-   Pluckthun, A. Bio/Technology 9: 545-551 (1991)-   Pozza, M., et al. (2000) J Rheumatol, 27(5): 1121-7.-   Priestley, J. V. et al. (2002) Can J Physiol Pharmacol, 80(5):    495-505.-   Qiao, L. Y. and Vizzard, M. A. (2002) J Comp Neurol, 454(2): 200-11.-   Ramer, M. S. et al. (1998) Neurosci Lett, 251(1): 53-6.-   Ramer, M. S. et al. (1999) Pain, Suppl 6: S111-20.-   Reiter, Y. et al, Nature Biotech, 14, 1239-1245, 1996-   Ridgeway, J. B. B. et al, Protein Eng., 9, 616-621, 1996-   Ro, L. S. et al. (1999) Pain, 79(2-3): 264-74.-   Robinson, J. R. ed., Sustained and Controlled Release Drug Delivery    Systems, Marcel Dekker, Inc., New York, 1978-   Sah, D. W. et al. (2003) Nat Rev Drug Discov, 2(6): 460-72.-   Sambrook and Russell, Molecular Cloning: a Laboratory Manual: 3rd    edition, 2001, Cold Spring Harbor Laboratory Press-   Sammons, M. J. et al. (2000) Brain Res, 876(1-2): 48-54.-   Schier et al., 1996, J. Mol. Biol. 263:551-567-   Stemmer, Nature, 1994, 370:389-391-   Vaughan et al., Nature Biotechnology 14 309-314, 1996.-   Voet & Voet, Biochemistry, 2nd Edition, (Wiley) 1995.-   Ward, E. S. et al., Nature 341, 544-546 (1989)-   Wess, L. In: BioCentury, The Bernstein Report on BioBusiness,    12(42), A1-A7, 2004.-   Whitelegg, N. R. u. and Rees, A. R (2000). Prot. Eng., 12, 815-824-   Witten, Ian H. & Frank, Eibe. Data Mining: Practical Machine    Learning Tools and Techniques with Java Implementations. Morgan    Kaufmann; (Oct. 11, 1999), ISBN: 1558605525-   Wold, et al. Multivariate data analysis in chemistry.    Chemometrics—Mathematics and Statistics in Chemistry (Ed.: B.    Kowalski), D. Reidel Publishing Company, Dordrecht, Holland, 1984    (ISBN 90-277-1846-6)-   Woolf, C. J. (1996) Philos Trans R Soc Lond B Biol Sci, 351(1338):    441-8.-   Yasuda, H. et al. (2003) Prog Neurobiol, 69(4): 229-85.

Zhang Y H, Nicol G D. Neurosci Lett. 2004 Aug. 12; 366(2):187-92. TABLE1 Neutralising potency of human IgG4 NGF antibodies in the calciummobilisation assay Human NGF Rat NGF Antibody IC₅₀ (nM) IC₅₀ (nM) Mab2560.76 ± 0.15^(a) 0.79 ± 0.08^(b) 1064F8  2.5 ± 0.6^(c)   5.5 1022E3   18± 6^(c) 14 1083H4  30  61^(e) 1021E5  76  75^(e) 1033G9   55 ± 20^(c) 261016A8   65 ± 12^(b) 14 1028F8  73 ND 1033B2  85 ND 1024C4  410 ±120*^(c) 565* 1057F11 3700* NDData indicate mean of two separate determinations, except ^(a)n = 14,^(b)n = 12, ^(c)n = 3, ^(d)n = 4 (mean ± sem) and ^(e)n = 1.*Values determined by extrapolation.ND = not determined.

TABLE 2a CDRs of 1021E5-derived optimised clones containing the HCDR3sequence LNPSLTA (SEQ ID NO: 531) HCDR1 HCDR2 HCDR3 Kabat numbering 3132 33 34 35 50 51 52 52A 53 54 55 56 57 58 59 60 61 62 63 64 65 95 96 9798 99 100 100A 100B 100C 100D 100E 100F 100G 100H LOT1021E05 T Y G I S GI I P I F D T G N S A Q S F Q G S S R I Y D Y A G G D H Y Y LOT1131H02 LN P S L T A LOT1132H09 L N P S L T A LOT1133C11 L N P S L T A LOT1134D09L N P S L T A LOT1146D07 L N P S L T A LOT1147A03 A L N P S L T ALOT1147D02 V L N P S L T A LOT1147F02 V N L N P S L T A LOT1147G09 A L NP S L T A LOT1149D09 L N P S L T A LOT1150D09 L N P S L T A LOT1150F01 DL N P S L T A LOT1150G08 L N P S L T A LOT1152D05 L N P S L T ALOT1152G10 V L N P S L T A LOT1156G06 S L N P S L T A LOT1160E03 L N P SL T A LOT1165D04 L N P S L T A LOT1215A06 N L N P S L T A LOT1218H05 S LN P S L T A LOT1219D09 L N P S L T A HCDR3 LCDR1 LCDR2 LCDR3 Kabatnumbering 100I 100J 100K 101 102 linker 24 25 26 27 27A 27B 28 29 30 3132 33 34 50 51 52 53 54 55 56 89 90 91 92 93 94 95 95A 95B 96 97LOT1021E05 Y D M D V S G S S S N I G N N Y V S D N N K R P S G T W D S SL S A W V LOT1131H02 T LOT1132H09 D T LOT1133C11 LOT1134D09 G LOT1146D07T LOT1147A03 G LOT1147D02 LOT1147F02 LOT1147G09 LOT1149D09 A LOT1150D09D LOT1150F01 LOT1150G08 G LOT1152D05 T LOT1152G10 P LOT1156G06LOT1160E03 N LOT1165D04 E LOT1215A06 LOT1218H05 T LOT1219D09 R scFv scFvIC50 in hu IC50 in rat NGF binding NGF binding Kabat numbering assay(nM) assay (nM) N LOT1021E05 94 269 5 LOT1131H02 <0.01 0.09 1 LOT1132H090.54 0.68 2 LOT1133C11 1.93 1.24 2 LOT1134D09 0.14 0.20 4 LOT1146D070.23 0.02 2 LOT1147A03 2.38 0.14 1 LOT1147D02 <0.01 0.10 1 LOT1147F020.76 1.04 2 LOT1147G09 0.25 0.01 2 LOT1149D09 0.01 0.12 1 LOT1150D090.70 0.14 1 LOT1150F01 0.20 0.16 2 LOT1150G08 0.20 0.54 1 LOT1152D050.67 0.06 1 LOT1152G10 0.23 0.05 2 LOT1156G06 0.32 1.05 1 LOT1160E030.14 0.22 1 LOT1165D04 0.11 0.29 1 LOT1215A06 1.43 0.06 1 LOT1218H05<0.01 0.03 1 LOT1219D09 0.06 0.11 1Columns on the right hand side of the table show an estimate of NGFneutralising potencies (IC50) for each clone. Purified scFv were assayedin an NGF-binding assay as described in Example 3.

TABLE 2b SEQ ID NOS corresponding to CDR sequences of clones shown inTable 2a HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 LOT1021E05 SEQ ID NO: 33SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40LOT1131H02 SEQ ID NO: 163 SEQ ID NO: 164 SEQ ID NO: 165 SEQ ID NO: 168SEQ ID NO: 169 SEQ ID NO: 170 LOT1132H09 SEQ ID NO: 183 SEQ ID NO: 184SEQ ID NO: 185 SEQ ID NO: 188 SEQ ID NO: 189 SEQ ID NO: 190 LOT1133C11SEQ ID NO: 193 SEQ ID NO: 194 SEQ ID NO: 195 SEQ ID NO: 198 SEQ ID NO:199 SEQ ID NO: 200 LOT1134D09 SEQ ID NO: 203 SEQ ID NO: 204 SEQ ID NO:205 SEQ ID NO: 208 SEQ ID NO: 209 SEQ ID NO: 210 LOT1146D07 SEQ ID NO:223 SEQ ID NO: 224 SEQ ID NO: 225 SEQ ID NO: 228 SEQ ID NO: 229 SEQ IDNO: 230 LOT1147A03 SEQ ID NO: 433 SEQ ID NO: 434 SEQ ID NO: 435 SEQ IDNO: 438 SEQ ID NO: 439 SEQ ID NO: 440 LOT1147D02 SEQ ID NO: 233 SEQ IDNO: 234 SEQ ID NO: 235 SEQ ID NO: 238 SEQ ID NO: 239 SEQ ID NO: 240LOT1147F02 SEQ ID NO: 443 SEQ ID NO: 444 SEQ ID NO: 445 SEQ ID NO: 448SEQ ID NO: 449 SEQ ID NO: 450 LOT1147G09 SEQ ID NO: 243 SEQ ID NO: 244SEQ ID NO: 245 SEQ ID NO: 248 SEQ ID NO: 249 SEQ ID NO: 250 LOT1149D09SEQ ID NO: 453 SEQ ID NO: 454 SEQ ID NO: 455 SEQ ID NO: 458 SEQ ID NO:459 SEQ ID NO: 460 LOT1150D09 SEQ ID NO: 463 SEQ ID NO: 464 SEQ ID NO:465 SEQ ID NO: 468 SEQ ID NO: 469 SEQ ID NO: 470 LOT1150F01 SEQ ID NO:253 SEQ ID NO: 254 SEQ ID NO: 255 SEQ ID NO: 258 SEQ ID NO: 259 SEQ IDNO: 260 LOT1150G08 SEQ ID NO: 473 SEQ ID NO: 474 SEQ ID NO: 475 SEQ IDNO: 478 SEQ ID NO: 479 SEQ ID NO: 480 LOT1152D05 SEQ ID NO: 483 SEQ IDNO: 484 SEQ ID NO: 485 SEQ ID NO: 488 SEQ ID NO: 489 SEQ ID NO: 490LOT1152G10 SEQ ID NO: 493 SEQ ID NO: 494 SEQ ID NO: 495 SEQ ID NO: 498SEQ ID NO: 499 SEQ ID NO: 500 LOT1156G06 SEQ ID NO: 503 SEQ ID NO: 504SEQ ID NO: 505 SEQ ID NO: 508 SEQ ID NO: 509 SEQ ID NO: 510 LOT1160E03SEQ ID NO: 293 SEQ ID NO: 294 SEQ ID NO: 295 SEQ ID NO: 298 SEQ ID NO:299 SEQ ID NO: 300 LOT1165D04 SEQ ID NO: 303 SEQ ID NO: 304 SEQ ID NO:305 SEQ ID NO: 308 SEQ ID NO: 309 SEQ ID NO: 310 LOT1215A06 SEQ ID NO:513 SEQ ID NO: 514 SEQ ID NO: 515 SEQ ID NO: 518 SEQ ID NO: 519 SEQ IDNO: 520 LOT1218H05 SEQ ID NO: 353 SEQ ID NO: 354 SEQ ID NO: 355 SEQ IDNO: 358 SEQ ID NO: 359 SEQ ID NO: 360 LOT1219D09 SEQ ID NO: 523 SEQ IDNO: 524 SEQ ID NO: 525 SEQ ID NO: 528 SEQ ID NO: 529 SEQ ID NO: 530

TABLE 3 NGF-neutralising potencies of optimised human IgG4 antibodies inthree assays of NGF function in whole cells FLIPR calcium mobilisationPC12 cell survival TF-1 cell proliferation IgG IC₅₀ (nM) IC₅₀ (nM) IC₅₀(nM) Clone Parent Human NGF Rat NGF Mouse NGF Human NGF Rat NGF HumanNGF Rat NGF Mouse NGF 1021E5 — 76^(a)   75 47 1300^(j)    1083H4 —30^(a)   61 54 1100^(j)    1126F1 1021E5 0.35 0.15 0.22 2.9  1126G51021E5 0.23 0.16 0.16 1.50 1126H5 1021E5 0.38 0.15 0.23 8.7  1127D91021E5 0.40 0.22 0.21 0.57 1127F9 1021E5 0.36 0.14 0.20 0.59 1131D71021E5 117    37 71 670    1131H2 1021E5 0.27 0.11 0.12 0.58 1132A91021E5 0.39 0.25 0.33 0.90 1132H9 1021E5 0.35 0.13 0.16 0.55 1133C111021E5   0.45^(a) 0.30^(a) 0.28^(a) 0.53 ± 0.13^(b) 0.42 ± 0.07^(b) 0.10± 0.01^(d) 0.12 ± 0.02^(d) 0.10 ± 0.02^(d) 1134D9 1021E5   0.31^(a)0.14^(a) 0.16^(a) 0.54 1145D1 1021E5 0.36 0.17 0.24 0.66 1146D7 1021E50.38 0.33 0.31 0.48 1147D2 1021E5 0.36 0.21 0.24 0.51 1147G9 1021E5 0.30± 0.04^(b) 0.21 ± 0.02^(b) 0.23 ± 0.02^(b) 0.76 ± 0.06^(b) 0.55 ±0.02^(b) 1150F1 1021E5 0.32 0.15 0.19 0.47 1152H5 1021E5 0.22 ± 0.05^(b)0.21 ± 0.05^(b) 0.14 ± 0.01^(b) 0.40 ± 0.04^(e) 0.38 ± 0.04^(e) 0.26 ±0.25^(d) 0.08 ± 0.0^(d)  0.07 ± 0.0^(d)  1155H1 1021E5 0.35 0.18 0.180.59 G1152H5 1152H5 0.22 0.27 0.18 1.1 ± 0.1^(b)  7.3 ± 0.9^(b) 0.44 ±0.17^(d) 1.44 ± 0.27^(d) 0.99 ± 0.29^(d) 1158A1 1021E5 0.34 0.12 0.110.48 1160E3 1021E5 0.33 0.13 0.12 0.40 1165D4 1021E5 0.26 ± 0.02^(b)0.15 ± 0.01^(b) 0.16 ± 0.04^(b) 0.41 ± 0.08^(e) 0.41 ± 0.06^(e) G1165D41165D4   0.32^(a) 0.33^(a) 0.27^(a) 0.86 ± 0.04^(b) 2.63 ± 0.03^(b)1175H8 1021E5 0.37 0.15 0.16 1.1  1211G10 1021E5 0.37 0.16 0.15 0.581214A1 1021E5   0.26^(a) 0.16^(a) 0.14^(a) 0.52 ± 0.07^(b) 0.43 ±0.07^(b) 1214D10 1021E5 0.29 0.14 0.13 0.35 1218H5 1021E5 0.33 0.13 0.150.47 1227H8 1083H4   0.44^(a) 0.43^(a) 0.48^(a) 0.70 ± 0.15^(b)   35 ±10^(b) 1230D8 1083H4   0.31^(a) 0.29^(a) 0.37^(a) 0.71 ± 0.14^(b)   37 ±4^(b)  1230H7 1021E5 0.27 ± 0.04^(b) 0.18 ± 0.03^(b) 0.15 ± 0.02^(b)0.42 ± 0.10^(c) 0.32 ± 0.05^(c) G1230H7 1230H7   0.31^(a) 0.34^(a)0.25^(a) 2.5 ± 0.2^(b)  7.5 ± 0.4^(b) 1252A5 1133C11 0.33 ± 0.03^(c)0.29 ± 0.06^(c) 0.26 ± 0.01^(c) 0.94 ± 0.13^(f )  2.7 ± 0.6^(f) 0.42 ±.15^(d)  0.72 ± 0.40^(d) 0.55 ± 0.32^(d) Mab 256 — 0.76 ± 0.15^(h) 0.79± 0.08^(g) 1.4 ± 0.2^(i ) 23 ± 1^(i )    44 ± 7^(f)  6 ± 4^(d) 5 ± 3^(d)7 ± 3^(d)Data are n = 1 except;^(a)n = 2;^(b)n = 3;^(c)n = 4;^(d)n = 5;^(e)n = 6;^(f)n = 7;^(g)n = 12;^(h)n = 14;^(i)n = 15;^(j)extrapolated

TABLE 4 Cross-reactivity of optimised human IgG4 NGF antibodies withother neurotrophins IgG BDNF, % NT-3, % NT-4, % 1133C11 0.7-3.1  0.9-1.8   0-1.7 1147G9 0-1.3 0.1-0.9   0-1.3 1152H5 0-1.5 0-0.50.2-1.2   1165D4 0-1.3 0-0.7 0-1.5 1214A1 0-1.4 0-1.0 0-1.0 1230H7 0-1.10-0.8 0-0.7 1252A5 0-1.4 0-1.1 0-0.9Data in columns show the range of calculated antibodycross-reactivities. Values are calculated as percentage of the signalobserved against each neurotrophin, relative to the NGF binding signalat the same test antibody concentration. Neurotrophins were coated toassay plates at a concentration of 100 ng/well, and binding of testantibodies was measured over the concentration range 0.03-10 nM. Datarepresent the result of a single experiment.

TABLE 5 Summary of binding inhibition constant determinations for 1252A5and G1152H5. Data represent mean ± s.e.m. of three independentexperiments. pKi vs human NGF pKi vs rat NGF IgG TrkA p75 TrkA p751252A5 10.26 ± 0.08* 9.85 ± 0.04 9.79 ± 0.04** 9.55 ± 0.03 G1152H5  9.59± 0.08^(§) 9.56 ± 0.04 9.18 ± 0.08^(§) 9.24 ± 0.05*P < 0.01 c.f. hu NGF/p75 interaction**P < 0.05 c.f. rat NGF/p75 interaction^(§)N/S c.f. p75Student's unpaired T-test

TABLE 6 IgG IC₅₀ (nM) Ki (nM) NGF 2.4 2.1 1252A5 0.068 0.061 G1152H50.204 0.184 MAB256 1.94 1.76 MAB5260Z 0.368 0.333

TABLE 7 IgG IC₅₀ (nM) Ki (nM) NGF 1.3 1.2 1252A5 0.151 0.140 G1152H50.538 0.499 MAB256 1.48 1.38 MAB5260Z 0.310 0.288

TABLE 8 IgG IC₅₀ (nM) Ki (nM) NGF 0.686 0.568 1252A5 0.188 0.155 G1152H50.348 0.288 MAB256 0.304 0.252 MAB5260Z 0.570 0.472

TABLE 9 IgG IC₅₀ (nM) Ki (nM) NGF 0.783 0.710 1252A5 0.302 0.274 G1152H50.781 0.708 MAB256 2.94 2.67 MAB5260Z 0.587 0.532

1. An isolated specific binding member for nerve growth factor (NGF),comprising an antibody antigen-binding site which is composed of a humanantibody VH domain and a human antibody VL domain and which comprises aset of CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the VHdomain comprises HCDR 1, HCDR2, HCDR3 and a framework and the VL domaincomprises LCDR1, LCDR2, LCDR3 and a framework, wherein the set of CDRsconsists of a set of CDRs selected from the group consisting of: the1133C11 set of CDRs, defined wherein the HCDR1 has the amino acidsequence of SEQ ID NO: 193, the HCDR2 has the amino acid sequence of SEQID NO: 194, the HCDR3 has the amino acid sequence of SEQ ID NO: 195, theLCDR1 has the amino acid sequence of SEQ ID NO: 198, the LCDR2 has theamino acid sequence of SEQ ID NO: 199, and the LCDR3 has the amino acidsequence of SEQ ID NO: 200; a set of CDRs which contains one or twoamino acid substitutions compared with the 1133C11 set of CDRs; each setof CDRs as shown for individual clones in Table 2; and a set of CDRswhich contains the 113C11 set of CDRs with the amino acid sequenceMISSLQP (SEQ ID NO: 533) or the amino acid sequence FNSALIS (SEQ ID NO:532) substituted for the amino acid sequence LNPSLTA (SEQ ID NO: 531)within HCDR3.
 2. An isolated specific binding member according to claim1 wherein the one or two substitutions are at one or two of thefollowing residues within the CDRs, using the standard numbering ofKabat: 31, 34 in HCDR1 51, 55, 56, 57, 58, 65 in HCDR2 96 in HCDR3 26,27, 27A, 27B, 28, 29, 30 in LCDR1 56 in LCDR2 90, 94 in LCDR3.
 3. Anisolated specific binding member according to claim 2 wherein the one ortwo substitutions are made at the following positions from among theidentified groups of possible substitute residues for each position:Substitute Residue selected from the Position of group consistingsubstitution of: 31 in HCDR1: A 34 in HCDR1: V 51 in HCDR2: V 55 inHCDR2: N 56 in HCDR2: A 57 in HCDR2: V 58 in HCDR2: S 65 in HCDR2: D 96in HCDR3: N 26 in LCDR1: T 26 in LCDR1: G 27 in LCDR1: N 27 in LCDR1: R27A in LCDR1: T 27A in LCDR1: P 27B in LCDR1: D 28 in LCDR1: T 29 inLCDR1: E 30 in LCDR1: D 56 in LCDR2: T 90 in LCDR3: A 94 in LCDR3: G.


4. An isolated specific binding member according to claim 3 whereinresidue 29 within LCDR1 is E.
 5. An isolated specific binding memberaccording to claim 1 comprising a set of CDRs which contains the 113C11set of CDRs with the amino acid sequence MISSLQP (SEQ ID NO: 533)substituted for the amino acid sequence LNPSLTA (SEQ ID NO: 531) withinHCDR3.
 6. An isolated specific binding member according to claim 1comprising a set of CDRs which contains the 113C11 set of CDRs with theamino acid sequence FNSALIS (SEQ ID NO: 532) substituted for the aminoacid sequence LNPSLTA (SEQ ID NO: 531) within HCDR3.
 7. An isolatedspecific binding member according to claim 1 comprising the 1133C11 setof CDRs.
 8. An isolated specific binding member according to claim 1wherein the VH domain framework is human heavy chain germline frameworkand/or the VL domain framework is human light chain germline framework.9. An isolated specific binding member according to claim 8 wherein theheavy chain germline framework comprises VH1 DP10.
 10. An isolatedspecific binding member according to claim 8 wherein the light chaingermline framework comprises VL Vλ1.
 11. An isolated specific bindingmember according to claim 1 which binds rat or mouse NGF.
 12. Anisolated specific binding member according to claim 1 whichpreferentially blocks NGF binding to TrkA receptor over NGF binding top75 receptor.
 13. A specific binding member according to claim 9comprising the 1252A5 VH domain (SEQ ID NO: 392).
 14. A specific bindingmember according to claim 9 comprising the 1252A5 VL domain (SEQ ID NO:397).
 15. A specific binding member according to claim 1 that bindshuman NGF with affinity equal to or better than the affinity of anantigen-binding site for human NGF formed by the 1252A5 VH domain (SEQID NO: 392) and the 1252A5 VL domain (SEQ ID NO: 397), the affinity ofthe specific binding member and the affinity of the antigen-binding sitebeing as determined under the same conditions.
 16. A specific bindingmember according to claim 1 that binds to and/or neutralises human NGF.17. A specific binding member according to claim 16 that neutralizeshuman NGF, with a potency equal to or better than the potency of a NGFantigen-binding site formed by the 1252A5 VH domain (SEQ ID NO: 392) andthe 1252A5 VL domain (SEQ ID NO: 397), the potency of the specificbinding member and the potency of the antigen-binding site being asdetermined under the same conditions.
 18. A specific binding memberaccording claim 9 comprising the 1152H5 VH domain (SEQ ID NO: 262). 19.A specific binding member according to claim 9 comprising the 1152H5 VLdomain (SEQ ID NO: 267).
 20. A specific binding member according claim 9comprising the 1165D4 VH domain (SEQ ID NO: 302).
 21. A specific bindingmember according to claim 9 comprising the 1165D4 VL domain (SEQ ID NO:307).
 22. A specific binding member according to claim 9 comprising the1230H7 VH domain (SEQ ID NO: 362).
 23. A specific binding memberaccording to claim 9 comprising the 1230H7 VL domain (SEQ ID NO: 367).24. A specific binding member according to claim 1 that comprises anscFv antibody molecule.
 25. A specific binding member according to claim1 that comprises an antibody constant region.
 26. A specific bindingmember according to claim 25 that comprises a whole antibody.
 27. Aspecific binding member according to claim 26 wherein the whole antibodyis IgG4.
 28. An isolated antibody VH domain of a specific binding memberaccording to claim
 1. 29. An isolated antibody VL domain of a specificbinding member according to claim
 1. 30. A composition comprising aspecific binding member, antibody VH domain or antibody VL according toclaim 1 and at least one additional component.
 31. A compositionaccording to claim 30 comprising a pharmaceutically acceptableexcipient, vehicle or carrier.
 32. An isolated nucleic acid whichcomprises a nucleotide sequence encoding a specific binding member orantibody VH or VL domain of a specific binding member according toclaim
 1. 33. A host cell in vitro transformed with nucleic acidaccording to claim
 32. 34. A method of producing a specific bindingmember or antibody VH or VL domain, the method comprising culturing hostcells according to claim 33 under conditions for production of saidspecific binding member or antibody VH or VL domain.
 35. A methodaccording to claim 34 further comprising isolating and/or purifying saidspecific binding member or antibody VH or VL variable domain.
 36. Amethod according to claim 34 further comprising formulating the specificbinding member or antibody VH or VL variable domain into a compositionincluding at least one additional component.
 37. A method for producingan antibody antigen-binding domain for NGF, the method comprisingproviding, by way of addition, deletion, substitution or insertion ofone or more amino acids in the amino acid sequence of a parent VH domaincomprising HCDR 1, HCDR2 and HCDR3, wherein the parent VH domain HCDR1,HCDR2 and HCDR3 are the 1252A5 set of HCDRs, defined wherein the HCDR1has the amino acid sequence of SEQ ID NO: 393, the HCDR2 has the aminoacid sequence of SEQ ID NO: 394, the HCDR3 has the amino acid sequenceof SEQ ID NO: 395, or the 1021E5 set of HCDRs, defined wherein the HCDR1has the amino acid sequence of SEQ ID NO: 33, the HCDR2 has the aminoacid sequence of SEQ ID NO: 34, the HCDR3 has the amino acid sequence ofSEQ ID NO: 35, a VH domain which is an amino acid sequence variant ofthe parent VH domain, and optionally combining the VH domain thusprovided with one or more VL domains to provide one or more VH/VLcombinations; and testing said VH domain which is an amino acid sequencevariant of the parent VH domain or the VH/VL combination or combinationsto identify an antibody antigen binding domain specific for NGF.
 38. Amethod according to claim 37 wherein the parent VH domain amino acidsequence is selected from the group consisting of SEQ ID NO: 392 and SEQID NO:
 32. 39. A method according to claim 37 wherein said one or moreVL domains is provided by way of addition, deletion, substitution orinsertion of one or more amino acids in the amino acid sequence of aparent VL domain comprising LCDR 1, LCDR2 and LCDR3, wherein the parentVL domain LCDR1, LCDR2 and LCDR3 are the 1252A5 set of LCDRs, definedwherein the LCDR1 has the amino acid sequence of SEQ ID NO: 398, theLCDR2 has the amino acid sequence of SEQ ID NO: 399, the LCDR3 has theamino acid sequence of SEQ ID NO: 400, or the 1021E5 set of LCDRs,defined wherein the LCDR1 has the amino acid sequence of SEQ ID NO: 38,the LCDR2 has the amino acid sequence of SEQ ID NO: 39, the LCDR3 hasthe amino acid sequence of SEQ ID NO: 40, producing one or more VLdomains each of which is an amino acid sequence variant of the parent VLdomain.
 40. A method according to claim 39 wherein the parent VL domainamino acid sequence is selected from the group consisting of SEQ ID NO:397 and SEQ ID NO:
 37. 41. A method according to claim 37, wherein theNGF is human NGF.
 42. A method for producing an antibody antigen-bindingdomain for NGF, the method comprising providing, by way of addition,deletion, substitution or insertion of one or more amino acids in theamino acid sequence of a parent VH domain comprising HCDR 1, HCDR2 andHCDR3, wherein the parent VH domain HCDR1, HCDR2 and HCDR3 are the1152H5 set of HCDRs, defined wherein the HCDR1 has the amino acidsequence of SEQ ID NO: 263, the HCDR2 has the amino acid sequence of SEQID NO: 264, the HCDR3 has the amino acid sequence of SEQ ID NO: 265, the1165D4 set of HCDRs, defined wherein the HCDR1 has the amino acidsequence of SEQ ID NO: 303, the HCDR2 has the amino acid sequence of SEQID NO: 304, the HCDR3 has the amino acid sequence of SEQ ID NO: 305, orthe 1230H7 set of HCDRs, defined wherein the HCDR1 has the amino acidsequence of SEQ ID NO: 363, the HCDR2 has the amino acid sequence of SEQID NO: 364, the HCDR3 has the amino acid sequence of SEQ ID NO: 365, aVH domain which is an amino acid sequence variant of the parent VHdomain, and optionally combining the VH domain thus provided with one ormore VL domains to provide one or more VH/VL combinations; and testingsaid VH domain which is an amino acid sequence variant of the parent VHdomain or the VH/VL combination or combinations to identify an antibodyantigen binding domain specific for NGF.
 43. A method according to claim42 wherein the parent VH domain amino acid sequence is selected from thegroup consisting of SEQ ID NO: 262, SEQ ID NO: 302 and SEQ ID NO: 362.44. A method according to claim 42 wherein said one or more VL domainsis provided by way of addition, deletion, substitution or insertion ofone or more amino acids in the amino acid sequence of a parent VL domaincomprising LCDR 1, LCDR2 and LCDR3, wherein the parent VL domain LCDR1,LCDR2 and LCDR3 are the 1152H5 set of LCDRs, defined wherein the LCDR1has the amino acid sequence of SEQ ID NO: 268, the LCDR2 has the aminoacid sequence of SEQ ID NO: 269, the LCDR3 has the amino acid sequenceof SEQ ID NO: 270, the 1165D4 set of LCDRs, defined wherein the LCDR1has the amino acid sequence of SEQ ID NO: 308, the LCDR2 has the aminoacid sequence of SEQ ID NO: 309, the LCDR3 has the amino acid sequenceof SEQ ID NO: 310, or the 1230H7 set of LCDRs, defined wherein the LCDR1has the amino acid sequence of SEQ ID NO: 368, the LCDR2 has the aminoacid sequence of SEQ ID NO: 369, the LCDR3 has the amino acid sequenceof SEQ ID NO: 370, producing one or more VL domains each of which is anamino acid sequence variant of the parent VL domain.
 45. A methodaccording to claim 44 wherein the parent VL domain amino acid sequenceis selected from the group consisting of SEQ ID NO: 267, SEQ ID NO: 307and SEQ ID NO:
 367. 46. A method according to claim 42, wherein the NGFis human NGF.
 47. A method according to claim 37 wherein said VH domainwhich is an amino acid sequence variant of the parent VH domain isprovided by CDR mutagenesis.
 48. A method according to claim 37 furthercomprising producing the antibody antigen-binding site as a component ofan IgG, scFv or Fab antibody molecule.
 49. A method for producing aspecific binding member that binds NGF, which method comprises:providing starting nucleic acid encoding a VH domain or a startingrepertoire of nucleic acids each encoding a VH domain, wherein the VHdomain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to bereplaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region; combiningsaid starting nucleic acid or starting repertoire with donor nucleicacid or donor nucleic acids encoding or produced by mutation of theamino acid sequence of HCDR1 SEQ ID NO: 193, HCDR2 SEQ ID NO: 194,and/or HCDR3 SEQ ID NO: 395, 265, 305, 365 or 35 such that said donornucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2and/or CDR3 region in the starting nucleic acid or starting repertoire,so as to provide a product repertoire of nucleic acids encoding VHdomains; expressing the nucleic acids of said product repertoire toproduce product VH domains; optionally combining said product VH domainswith one or more VL domains; selecting a specific binding member forNGF, which specific binding member comprises a product VH domain andoptionally a VL domain; and recovering said specific binding member ornucleic acid encoding it.
 50. A method according to claim 49 wherein thedonor nucleic acids are produced by mutation of said HCDR1 and/or HCDR2.51. A method according to claim 49 wherein the donor nucleic acid isproduced by mutation of HCDR3.
 52. A method according to claim 51comprising providing the donor nucleic acid by mutation of nucleic acidencoding the amino acid sequence of 1252A5 HCDR3 (SEQ ID NO: 395) or1021E5 HCDR3 (SEQ ID NO: 35).
 53. A method according to claim 49comprising providing the donor nucleic acid by random mutation ofnucleic acid.
 54. A method according to claim 49 further comprisingattaching a product VH domain that is comprised within the recoveredspecific binding member to an antibody constant region.
 55. A methodaccording to claim 49 comprising providing an IgG, scFv or Fab antibodymolecule comprising the product VH domain and a VL domain.
 56. A methodaccording to claim 49, wherein the NGF is human NGF.
 57. A methodaccording to claim 37, further comprising testing the antibodyantigen-binding domain or specific binding member that binds NGF forability to neutralize NGF.
 58. A method according to claim 57 wherein aspecific binding member that comprises an antibody fragment that bindsand neutralizes NGF is obtained.
 59. A method according to claim 58wherein the antibody fragment is an scFv antibody molecule.
 60. A methodaccording to claim 58 wherein the antibody fragment is an Fab antibodymolecule.
 61. A method according to claim 59 further comprisingproviding the VH domain and/or the VL domain of the antibody fragment ina whole antibody.
 62. A method according to claim 37 further comprisingformulating the specific binding member that binds NGF, antibodyantigen-binding site or an antibody VH or VL variable domain of thespecific binding member or antibody antigen-binding site that binds NGF,into a composition including at least one additional component.
 63. Amethod according to claim 37 further comprising binding a specificbinding member that binds NGF to NGF or a fragment of NGF.
 64. A methodcomprising binding a specific binding member that binds NGF according toclaim 1 to human NGF or a fragment of human NGF.
 65. A method accordingto claim 63 wherein said binding takes place in vitro.
 66. A methodaccording to claim 63, comprising binding the specific binding member tohuman NGF or a fragment of human NGF.
 67. A method according to claim 63comprising determining the amount of binding of specific binding memberto NGF or a fragment of NGF.
 68. A method according to claim 37 furthercomprising use of the specific binding member in the manufacture of amedicament for treatment of a disease or disorder in which NGF plays arole.
 69. Use of a specific binding member according to claim 1 in themanufacture of a medicament for treatment of a disease or disorder inwhich NGF plays a role.
 70. A method of treatment of a disease ordisorder in which NGF plays a role, the method comprising administeringa specific binding member according to claim 1 to a patient with thedisease or disorder or at risk of developing the disease or disorder.71. A method or use according to claim 68, wherein the disease ordisorder is selected from the group consisting of pain, asthma, chronicobstructive pulmonary disease, pulmonary fibrosis, other diseases ofairway inflammation, diabetic neuropathy, cardiac arrhythmias, HIV,arthritis, psoriasis and cancer.
 72. A method or use according to claim71 wherein said treatment is of pain.